Advertisement

CXCL9 and CXCL10 are differentially associated with systemic organ involvement and pulmonary disease severity in sarcoidosis

Open ArchivePublished:November 20, 2019DOI:https://doi.org/10.1016/j.rmed.2019.105822

      Highlights

      • Serum CXCL10 was negatively associated with FVC, DLCO, and TLC in sarcoidosis subjects.
      • Subjects with increased levels of CXCL10 had increased risk of PFT declines.
      • Serum CXCL9 positively correlated with organ involvement.
      • CXCL10 gene expression and blood monocyte levels positively correlated with CXCL10 protein levels, whereas CXCL9 did not.
      • Both CXCL9 and CXCL10 were lower with higher immunosuppression usage.

      Abstract

      Background

      Sarcoidosis is a granulomatous inflammatory disease with limited blood markers to predict outcomes. The interferon-gamma (IFN-γ)-inducible chemotactic cytokines (chemokines), CXCL9 and CXCL10, are both increased in sarcoidosis patients, yet they possess important molecular differences. Our study determined if serum chemokines correlated with different aspects of disease severity.

      Methods

      We measured CXCL9 and CXCL10 serum levels at initial study visits and longitudinally in sarcoidosis subjects using ELISA. We examined these chemokines’ relationships with pulmonary and organ involvement outcomes, their gene expression, peripheral blood immune cell populations, and immunosuppression use.

      Results

      Higher CXCL10 levels negatively correlated with FVC, TLC, and DLCO at subjects’ initial visit and when measured repeatedly over two years. CXCL10 also positively correlated with longitudinal respiratory symptom severity. Additionally, for every log10(CXCL10) increase, the risk of longitudinal pulmonary function decline increased 8.8 times over the 5-year study period (95% CI 1.6–50, p = 0.014, log10(CXCL0) range 0.84–2.7). In contrast, CXCL9 levels positively correlated with systemic organ involvement at initial study visit (1.5 additional organs involved for every log10(CXCL9) increase, 95% CI 1.1–2.0, p = 0.022, log10(CXCL9) range 1.3–3.3). CXCL10, not CXCL9, positively correlated with its own blood gene expression and monocyte level. Immunosuppressive treatment was associated with lower levels of both chemokines.

      Conclusions

      In sarcoidosis subjects, serum CXCL9 levels correlated with systemic organ involvement and CXCL10 levels strongly correlated with respiratory outcomes, which may ultimately prove helpful in clinical management. These differing associations may be due to differences in cellular regulation and tissue origin.

      Keywords

      Abbreviations

      ANOVA
      Analysis of Variance
      DLCO
      Diffusing Capacity of the Lungs for Carbon Monoxide
      DMARD
      Disease Modifying Antirheumatic Drug
      ELISA
      Enzyme-Linked Immunosorbent Assay
      FEV1
      Forced Expiratory Volume in 1 Second
      FVC
      Forced Vital Capacity
      FEV1/FVC
      Forced Expiratory Volume in 1 Second to Forced Vital Capacity ratio
      HR
      Hazard Ratio
      IFN-α
      Interferon-alpha
      IFN-α2a
      Interferon-alpha 2a
      IFN-γ
      Interferon-Gamma
      LPS
      Lipopolysaccharide
      OR
      Odds Ratio
      %pred
      Percent Predicted
      PET
      Positron Emission Tomography
      TLC
      Total Lung Capacity
      TNF-α
      Tumor Necrosis Factor Alpha
      UCSF
      University of California, San Francisco

      1. Introduction

      Sarcoidosis is a systemic inflammatory disease characterized by non-necrotizing granulomas occurring in adults of any age, sex, or race [
      • Chappell A.G.
      • Cheung W.Y.
      • Hutchings H.A.
      Sarcoidosis: a long-term follow up study. Sarcoidosis, vasculitis, and diffuse lung diseases.
      ,
      • Iannuzzi M.C.
      • Rybicki B.A.
      • Teirstein A.S.
      Sarcoidosis.
      ,
      • Rabin D.L.
      • Richardson M.S.
      • Stein S.R.
      • Yeager Jr., H.
      Sarcoidosis severity and socioeconomic status.
      ]. Although any organ can be affected, pulmonary involvement occurs in over 90% of patients, which can lead to abnormal lung function and debilitating respiratory symptoms [
      • Hunninghake G.W.
      • Costabel U.
      • Ando M.
      • Baughman R.
      • Cordier J.F.
      • du Bois R.
      • Eklund A.
      • Kitaichi M.
      • Lynch J.
      • Rizzato G.
      • Rose C.
      • Selroos O.
      • Semenzato G.
      • Sharma O.P.
      ATS/ERS/WASOG statement on sarcoidosis. American thoracic society/European respiratory society/world association of sarcoidosis and other granulomatous disorders. Sarcoidosis, vasculitis, and diffuse lung diseases.
      ]. Two important clinical challenges in the disease include predicting which patients will develop progressive pulmonary and multi-organ disease, and deciding who to treat and when to stop since treatment is not curative [
      • Gerke A.K.
      Morbidity and mortality in sarcoidosis.
      ,
      • Gerke A.K.
      • Judson M.A.
      • Cozier Y.C.
      • Culver D.A.
      • Koth L.L.
      Disease burden and variability in sarcoidosis.
      ,
      • Dwyer-Lindgren L.
      • Bertozzi-Villa A.
      • Stubbs R.W.
      • Morozoff C.
      • Shirude S.
      • Naghavi M.
      • Mokdad A.H.
      • Murray C.J.L.
      Trends and patterns of differences in chronic respiratory disease mortality among US counties.
      ]. These clinical challenges persist partly due to the paucity of detailed longitudinal sarcoidosis studies.
      IFN-gamma (IFN-γ) is one of the main cytokines in sarcoidosis-associated inflammation [
      • Robinson B.W.
      • McLemore T.L.
      • Crystal R.G.
      Gamma interferon is spontaneously released by alveolar macrophages and lung T lymphocytes in patients with pulmonary sarcoidosis.
      ,
      • Prasse A.
      • Georges C.G.
      • Biller H.
      • Hamm H.
      • Matthys H.
      • Luttmann W.
      • Virchow J.C.
      Th1 cytokine pattern in sarcoidosis is expressed by bronchoalveolar CD4(+) and CD8(+) T cells.
      ,
      • Inui N.
      • Chida K.
      • Suda T.
      • Nakamura H.
      TH1/TH2 and TC1/TC2 profiles in peripheral blood and bronchoalveolar lavage fluid cells in pulmonary sarcoidosis.
      ,
      • Mollers M.
      • Aries S.P.
      • Dromann D.
      • Mascher B.
      • Braun J.
      • Dalhoff K.
      Intracellular cytokine repertoire in different T cell subsets from patients with sarcoidosis.
      ,
      • Wahlstrom J.
      • Katchar K.
      • Wigzell H.
      • Olerup O.
      • Eklund A.
      • Grunewald J.
      Analysis of intracellular cytokines in CD4+ and CD8+ lung and blood T cells in sarcoidosis.
      ,
      • Kriegova E.
      • Fillerova R.
      • Tomankova T.
      • Hutyrova B.
      • Mrazek F.
      • Tichy T.
      • Kolek V.
      • du Bois R.M.
      • Petrek M.
      T-helper cell type-1 transcription factor T-bet is upregulated in pulmonary sarcoidosis.
      ], and several studies have identified upregulation of interferon-inducible chemotactic cytokines (chemokines) in the blood of sarcoidosis patients compared to control groups [
      • Antoniou K.M.
      • Tzouvelekis A.
      • Alexandrakis M.G.
      • Sfiridaki K.
      • Tsiligianni I.
      • Rachiotis G.
      • Tzanakis N.
      • Bouros D.
      • Milic-Emili J.
      • Siafakas N.M.
      Different angiogenic activity in pulmonary sarcoidosis and idiopathic pulmonary fibrosis.
      ,
      • Sugiyama K.
      • Mukae H.
      • Ishii H.
      • Kakugawa T.
      • Ishimoto H.
      • Nakayama S.
      • Shirai R.
      • Fujii T.
      • Mizuta Y.
      • Kohno S.
      Elevated levels of interferon gamma-inducible protein-10 and epithelial neutrophil-activating peptide-78 in patients with pulmonary sarcoidosis.
      ,
      • Nishioka Y.
      • Manabe K.
      • Kishi J.
      • Wang W.
      • Inayama M.
      • Azuma M.
      • Sone S.
      CXCL9 and 11 in patients with pulmonary sarcoidosis: a role of alveolar macrophages.
      ,
      • Nureki S.
      • Miyazaki E.
      • Ando M.
      • Ueno T.
      • Fukami T.
      • Kumamoto T.
      • Sugisaki K.
      • Tsuda T.
      Circulating levels of both Th1 and Th2 chemokines are elevated in patients with sarcoidosis.
      ,
      • Nagata K.
      • Maruyama K.
      • Uno K.
      • Shinomiya K.
      • Yoneda K.
      • Hamuro J.
      • Sugita S.
      • Yoshimura T.
      • Sonoda K.H.
      • Mochizuki M.
      • Kinoshita S.
      Simultaneous analysis of multiple cytokines in the vitreous of patients with sarcoid uveitis.
      ,
      • Takeuchi M.
      • Oh I.K.
      • Suzuki J.
      • Hattori T.
      • Takeuchi A.
      • Okunuki Y.
      • Usui Y.
      • Usui M.
      Elevated serum levels of CXCL9/monokine induced by interferon-gamma and CXCL10/interferon-gamma-inducible protein-10 in ocular sarcoidosis.
      ,
      • Koth L.L.
      • Solberg O.D.
      • Peng J.C.
      • Bhakta N.R.
      • Nguyen C.P.
      • Woodruff P.G.
      Sarcoidosis blood transcriptome reflects lung inflammation and overlaps with tuberculosis.
      ,
      • Su R.
      • Nguyen M.L.
      • Agarwal M.R.
      • Kirby C.
      • Nguyen C.P.
      • Ramstein J.
      • Darnell E.P.
      • Gomez A.D.
      • Ho M.
      • Woodruff P.G.
      • Koth L.L.
      Interferon-inducible chemokines reflect severity and progression in sarcoidosis.
      ,
      • Su R.
      • Li M.M.
      • Bhakta N.R.
      • Solberg O.D.
      • Darnell E.P.
      • Ramstein J.
      • Garudadri S.
      • Ho M.
      • Woodruff P.G.
      • Koth L.L.
      Longitudinal analysis of sarcoidosis blood transcriptomic signatures and disease outcomes.
      ,
      • Arger N.K.
      • Ho M.
      • Woodruff P.G.
      • Koth L.L.
      Serum CXCL11 correlates with pulmonary outcomes and disease burden in sarcoidosis.
      ]. These chemokines, CXCL9, CXCL10, and CXCL11 all bind to the CXCR3 receptor, and are responsible for homing of CD4+ T cells, monocytes, and other inflammatory cells to sites of inflammation including granulomas [
      • Nishioka Y.
      • Manabe K.
      • Kishi J.
      • Wang W.
      • Inayama M.
      • Azuma M.
      • Sone S.
      CXCL9 and 11 in patients with pulmonary sarcoidosis: a role of alveolar macrophages.
      ,
      • Nureki S.
      • Miyazaki E.
      • Ando M.
      • Ueno T.
      • Fukami T.
      • Kumamoto T.
      • Sugisaki K.
      • Tsuda T.
      Circulating levels of both Th1 and Th2 chemokines are elevated in patients with sarcoidosis.
      ,
      • Groom J.R.
      • Luster A.D.
      CXCR3 ligands: redundant, collaborative and antagonistic functions.
      ,
      • Grimm M.C.
      • Doe W.F.
      Chemokines in inflammatory bowel disease mucosa: expression of RANTES, macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, and gamma-interferon-inducible protein-10 by macrophages, lymphocytes, endothelial cells, and granulomas.
      ,
      • Kishi J.
      • Nishioka Y.
      • Kuwahara T.
      • Kakiuchi S.
      • Azuma M.
      • Aono Y.
      • Makino H.
      • Kinoshita K.
      • Kishi M.
      • Batmunkh R.
      • Uehara H.
      • Izumi K.
      • Sone S.
      Blockade of Th1 chemokine receptors ameliorates pulmonary granulomatosis in mice.
      ,
      • Aranday-Cortes E.
      • Bull N.C.
      • Villarreal-Ramos B.
      • Gough J.
      • Hicks D.
      • Ortiz-Pelaez A.
      • Vordermeier H.M.
      • Salguero F.J.
      Upregulation of IL-17A, CXCL9 and CXCL10 in early-stage granulomas induced by Mycobacterium bovis in cattle.
      ,
      • Torraca V.
      • Cui C.
      • Boland R.
      • Bebelman J.P.
      • van der Sar A.M.
      • Smit M.J.
      • Siderius M.
      • Spaink H.P.
      • Meijer A.H.
      The CXCR3-CXCL11 signaling axis mediates macrophage recruitment and dissemination of mycobacterial infection.
      ,
      • Agostini C.
      • Cassatella M.
      • Zambello R.
      • Trentin L.
      • Gasperini S.
      • Perin A.
      • Piazza F.
      • Siviero M.
      • Facco M.
      • Dziejman M.
      • Chilosi M.
      • Qin S.
      • Luster A.D.
      • Semenzato G.
      Involvement of the IP-10 chemokine in sarcoid granulomatous reactions.
      ]. These chemokines also participate in other functions related to angiogenesis and cell proliferation [
      • Aksoy M.O.
      • Yang Y.
      • Ji R.
      • Reddy P.J.
      • Shahabuddin S.
      • Litvin J.
      • Rogers T.J.
      • Kelsen S.G.
      CXCR3 surface expression in human airway epithelial cells: cell cycle dependence and effect on cell proliferation.
      ,
      • Strieter R.M.
      • Burdick M.D.
      • Gomperts B.N.
      • Belperio J.A.
      • Keane M.P.
      CXC chemokines in angiogenesis.
      ,
      • Liu L.
      • Callahan M.K.
      • Huang D.
      • Ransohoff R.M.
      Chemokine receptor CXCR3: an unexpected enigma.
      ]. Results from studies in mice suggest that these chemokines have nonredundant functions in vivo [
      • Groom J.R.
      • Luster A.D.
      CXCR3 ligands: redundant, collaborative and antagonistic functions.
      ,
      • Hancock W.W.
      • Gao W.
      • Csizmadia V.
      • Faia K.L.
      • Shemmeri N.
      • Luster A.D.
      Donor-derived IP-10 initiates development of acute allograft rejection.
      ]. For example, CXCL11 has unique properties related to T cell function and can also bind to the CXCR7 receptor [
      • Burns J.M.
      • Summers B.C.
      • Wang Y.
      • Melikian A.
      • Berahovich R.
      • Miao Z.
      • Penfold M.E.
      • Sunshine M.J.
      • Littman D.R.
      • Kuo C.J.
      • Wei K.
      • McMaster B.E.
      • Wright K.
      • Howard M.C.
      • Schall T.J.
      A novel chemokine receptor for SDF-1 and I-TAC involved in cell survival, cell adhesion, and tumor development.
      ,
      • Zohar Y.
      • Wildbaum G.
      • Novak R.
      • Salzman A.L.
      • Thelen M.
      • Alon R.
      • Barsheshet Y.
      • Karp C.L.
      • Karin N.
      CXCL11-dependent induction of FOXP3-negative regulatory T cells suppresses autoimmune encephalomyelitis.
      ], which led us to examine it in a previous study where we found its relationships to clinical outcomes of lung function and organ involvement [
      • Arger N.K.
      • Ho M.
      • Woodruff P.G.
      • Koth L.L.
      Serum CXCL11 correlates with pulmonary outcomes and disease burden in sarcoidosis.
      ].
      The other two chemokines, CXCL9 and CXCL10, can also bind and signal through the CXCR3 receptor, but with different binding affinities [
      • Colvin R.A.
      • Campanella G.S.
      • Sun J.
      • Luster A.D.
      Intracellular domains of CXCR3 that mediate CXCL9, CXCL10, and CXCL11 function.
      ,
      • Cole K.E.
      • Strick C.A.
      • Paradis T.J.
      • Ogborne K.T.
      • Loetscher M.
      • Gladue R.P.
      • Lin W.
      • Boyd J.G.
      • Moser B.
      • Wood D.E.
      • Sahagan B.G.
      • Neote K.
      Interferon-inducible T cell alpha chemoattractant (I-TAC): a novel non-ELR CXC chemokine with potent activity on activated T cells through selective high affinity binding to CXCR3.
      ]. They also differ in how they are induced, their ability to antagonize other receptors [
      • Xanthou G.
      • Duchesnes C.E.
      • Williams T.J.
      • Pease J.E.
      CCR3 functional responses are regulated by both CXCR3 and its ligands CXCL9, CXCL10 and CXCL11.
      ], and their spatial expression in vivo. For example, CXCL9 is solely induced by IFN-γ, whereas CXCL10 can also be induced by TNF-α, IFN-α, and LPS [
      • Groom J.R.
      • Luster A.D.
      CXCR3 ligands: redundant, collaborative and antagonistic functions.
      ,
      • Wang Q.
      • Nagarkar D.R.
      • Bowman E.R.
      • Schneider D.
      • Gosangi B.
      • Lei J.
      • Zhao Y.
      • McHenry C.L.
      • Burgens R.V.
      • Miller D.J.
      • Sajjan U.
      • Hershenson M.B.
      Role of double-stranded RNA pattern recognition receptors in rhinovirus-induced airway epithelial cell responses.
      ,
      • Amichay D.
      • Gazzinelli R.T.
      • Karupiah G.
      • Moench T.R.
      • Sher A.
      • Farber J.M.
      Genes for chemokines MuMig and Crg-2 are induced in protozoan and viral infections in response to IFN-gamma with patterns of tissue expression that suggest nonredundant roles in vivo.
      ,
      • Nakanishi Y.
      • Lu B.
      • Gerard C.
      • Iwasaki A.
      CD8(+) T lymphocyte mobilization to virus-infected tissue requires CD4(+) T-cell help.
      ,
      • Medoff B.D.
      • Wain J.C.
      • Seung E.
      • Jackobek R.
      • Means T.K.
      • Ginns L.C.
      • Farber J.M.
      • Luster A.D.
      CXCR3 and its ligands in a murine model of obliterative bronchiolitis: regulation and function.
      ,
      • Ohmori Y.
      • Wyner L.
      • Narumi S.
      • Armstrong D.
      • Stoler M.
      • Hamilton T.A.
      Tumor necrosis factor-alpha induces cell type and tissue-specific expression of chemoattractant cytokines in vivo.
      ,
      • Ciesielski C.J.
      • Andreakos E.
      • Foxwell B.M.
      • Feldmann M.
      TNFalpha-induced macrophage chemokine secretion is more dependent on NF-kappaB expression than lipopolysaccharides-induced macrophage chemokine secretion.
      ,
      • Luster A.D.
      • Unkeless J.C.
      • Ravetch J.V.
      Gamma-interferon transcriptionally regulates an early-response gene containing homology to platelet proteins.
      ,
      • Proost P.
      • Verpoest S.
      • Van de Borne K.
      • Schutyser E.
      • Struyf S.
      • Put W.
      • Ronsse I.
      • Grillet B.
      • Opdenakker G.
      • Van Damme J.
      Synergistic induction of CXCL9 and CXCL11 by Toll-like receptor ligands and interferon-gamma in fibroblasts correlates with elevated levels of CXCR3 ligands in septic arthritis synovial fluids.
      ,
      • Proost P.
      • Vynckier A.K.
      • Mahieu F.
      • Put W.
      • Grillet B.
      • Struyf S.
      • Wuyts A.
      • Opdenakker G.
      • Van Damme J.
      Microbial Toll-like receptor ligands differentially regulate CXCL10/IP-10 expression in fibroblasts and mononuclear leukocytes in synergy with IFN-gamma and provide a mechanism for enhanced synovial chemokine levels in septic arthritis.
      ]. Furthermore, CXCL9 and CXCL10 are expressed in different tissues in humans [
      • Proost P.
      • Verpoest S.
      • Van de Borne K.
      • Schutyser E.
      • Struyf S.
      • Put W.
      • Ronsse I.
      • Grillet B.
      • Opdenakker G.
      • Van Damme J.
      Synergistic induction of CXCL9 and CXCL11 by Toll-like receptor ligands and interferon-gamma in fibroblasts correlates with elevated levels of CXCR3 ligands in septic arthritis synovial fluids.
      ,
      • Proost P.
      • Vynckier A.K.
      • Mahieu F.
      • Put W.
      • Grillet B.
      • Struyf S.
      • Wuyts A.
      • Opdenakker G.
      • Van Damme J.
      Microbial Toll-like receptor ligands differentially regulate CXCL10/IP-10 expression in fibroblasts and mononuclear leukocytes in synergy with IFN-gamma and provide a mechanism for enhanced synovial chemokine levels in septic arthritis.
      ], and are produced by different cellular sources in mouse models directly comparing their production in vivo [
      • Amichay D.
      • Gazzinelli R.T.
      • Karupiah G.
      • Moench T.R.
      • Sher A.
      • Farber J.M.
      Genes for chemokines MuMig and Crg-2 are induced in protozoan and viral infections in response to IFN-gamma with patterns of tissue expression that suggest nonredundant roles in vivo.
      ]. These prior observations led us to hypothesize that CXCL9 and CXCL10 may be associated with different clinical outcomes based on their differing biological properties. In this study, we wanted to determine if lung disease severity and multi-organ involvement are differentially influenced by circulating levels of CXCL9 and CXCL10. To understand the relationship between chemokine levels, pulmonary physiology, organ involvement, and other clinical data, we took advantage of longitudinal measurements obtained over a five-year follow-up period within our University of California, San Francisco (UCSF) Sarcoidosis Cohort [
      • Benn B.S.
      • Lehman Z.
      • Kidd S.A.
      • Ho M.
      • Sun S.
      • Ramstein J.
      • Arger N.K.
      • Nguyen C.P.
      • Su R.
      • Gomez A.
      • Gelfand J.M.
      • Koth L.L.
      Clinical and biological insights from the university of California san Francisco prospective and longitudinal cohort.
      ]. Finding unique relationships between chemokines and distinct clinical features may provide more specific markers for clinically meaningful outcomes to inform the care of sarcoidosis patients.

      2. Methods

      2.1 Study population and measurements

      We enrolled sarcoidosis subjects who met diagnostic criteria established by the American Thoracic Society [
      • Hunninghake G.W.
      • Costabel U.
      • Ando M.
      • Baughman R.
      • Cordier J.F.
      • du Bois R.
      • Eklund A.
      • Kitaichi M.
      • Lynch J.
      • Rizzato G.
      • Rose C.
      • Selroos O.
      • Semenzato G.
      • Sharma O.P.
      ATS/ERS/WASOG statement on sarcoidosis. American thoracic society/European respiratory society/world association of sarcoidosis and other granulomatous disorders. Sarcoidosis, vasculitis, and diffuse lung diseases.
      ] at any point in their disease course as part of the UCSF Sarcoidosis Cohort as previously described [
      • Benn B.S.
      • Lehman Z.
      • Kidd S.A.
      • Ho M.
      • Sun S.
      • Ramstein J.
      • Arger N.K.
      • Nguyen C.P.
      • Su R.
      • Gomez A.
      • Gelfand J.M.
      • Koth L.L.
      Clinical and biological insights from the university of California san Francisco prospective and longitudinal cohort.
      ]. The study design required tissue confirmation of granulomatous inflammation and no alternative lung disease at enrollment but individuals did not have to be newly diagnosed to participate. This cohort had follow-up visits every 6–12 months for up to 66 months (~5 years). At each visit, blood sampling was performed and clinical data were collected. The following data were used in this study: demographics, organ involvement at the initial visit (as assessed by physician review of medical records) [
      • Benn B.S.
      • Lehman Z.
      • Kidd S.A.
      • Ho M.
      • Sun S.
      • Ramstein J.
      • Arger N.K.
      • Nguyen C.P.
      • Su R.
      • Gomez A.
      • Gelfand J.M.
      • Koth L.L.
      Clinical and biological insights from the university of California san Francisco prospective and longitudinal cohort.
      ], chest X-ray imaging at the initial visit, clinical laboratory tests including complete blood counts, and pulmonary function tests, which included forced expiratory volume in 1 s (FEV1) percent predicted (%pred), forced vital capacity (FVC %pred), diffusing capacity for carbon monoxide (DLCO %pred), and total lung capacity (TLC %pred), and severity of respiratory symptoms as assessed by the UCSD Dyspnea Questionnaire [
      • Eakin E.G.
      • Resnikoff P.M.
      • Prewitt L.M.
      • Ries A.L.
      • Kaplan R.M.
      Validation of a new dyspnea measure: the UCSD shortness of breath questionnaire.
      ,
      • Swigris J.J.
      • Yorke J.
      • Sprunger D.B.
      • Swearingen C.
      • Pincus T.
      • du Bois R.M.
      • Brown K.K.
      • Fischer A.
      Assessing dyspnea and its impact on patients with connective tissue disease-related interstitial lung disease.
      ,
      • Swigris J.J.
      • Han M.
      • Vij R.
      • Noth I.
      • Eisenstein E.L.
      • Anstrom K.J.
      • Brown K.K.
      • Fairclough D.
      The UCSD shortness of breath questionnaire has longitudinal construct validity in idiopathic pulmonary fibrosis.
      ]. We obtained immunosuppression use history, including dosages of oral corticosteroids or disease-modifying antirheumatic drugs (DMARDs), specifically, methotrexate, azathioprine, mycophenolate, colchicine, hydroxychloroquine, or anti-TNF-α therapy that subjects were actively taking at the time of their study visits. For the current analysis, we included gene transcript levels of CXCL9 and CXCL10 from whole blood RNA samples that we previously described [
      • Su R.
      • Li M.M.
      • Bhakta N.R.
      • Solberg O.D.
      • Darnell E.P.
      • Ramstein J.
      • Garudadri S.
      • Ho M.
      • Woodruff P.G.
      • Koth L.L.
      Longitudinal analysis of sarcoidosis blood transcriptomic signatures and disease outcomes.
      ].

      2.2 Protein assay

      We measured levels of CXCL9 and CXCL10 in serum using Quantikine Colorimetric Sandwich ELISA kits on samples obtained over a two-year time period after enrollment per manufacturer's instructions (R&D Systems Minneapolis, MN, USA). Samples were thawed, analyzed in duplicate, and processed in one batch. The duplicates per sample were averaged for final interpretation.

      2.3 Statistical analysis

      We normalized chemokine levels and dyspnea scores using log10 transformations given their skewed distributions prior to analysis. We used Chi-squared tests for analyzing categorical variables, t-tests for bivariate comparisons of continuously distributed parametric data, and analysis of variance (ANOVA) to analyze variables with more than two groups. For adjusted cross-sectional analyses of data obtained at the initial visit, we used linear regression models for continuous and normally distributed clinical outcome variables; logistic regression for binary outcomes; and Poisson regression for count data (i.e. number of organs involved, where thoracic adenopathy and/or lung parenchymal involvement was considered one organ).
      To analyze all follow-up visit data and account for drop-outs, we used mixed effects linear regression models that assessed correlations between changes in predictors and dependent variables measured over multiple visits. The fixed effects were the clinical predictors of interest and the random effects were the subjects. In these mixed effects models, we made conservative assumptions by allowing each subject to have a separate intercept, allowing slopes to vary by subject, and using unstructured covariation matrices [
      • Vittinghoff E.
      • Glidden D.V.
      • Shiboski S.C.
      • McCulloch C.E.
      Statistics for Biology and Health.
      ,
      • McLean R.A.
      • Sanders W.L.
      • Stroup W.W.
      A unified approach to mixed linear models.
      ]. To determine if either chemokine was predictive of clinically significant pulmonary function decline [
      • Keir G.
      • Wells A.U.
      Assessing pulmonary disease and response to therapy: which test?.
      ,
      • Baughman R.P.
      • Teirstein A.S.
      • Judson M.A.
      • Rossman M.D.
      • Yeager Jr., H.
      • Bresnitz E.A.
      • DePalo L.
      • Hunninghake G.
      • Iannuzzi M.C.
      • Johns C.J.
      • McLennan G.
      • Moller D.R.
      • Newman L.S.
      • Rabin D.L.
      • Rose C.
      • Rybicki B.
      • Weinberger S.E.
      • Terrin M.L.
      • Knatterud G.L.
      • Cherniak R.
      Clinical characteristics of patients in a case control study of sarcoidosis.
      ,
      • Schoenfeld D.
      Partial residuals for the proportional hazards regression model.
      ], we performed a time-to-event analysis as described in the Supplementary Materials.
      To identify independent predictors of chemokine levels, we performed similar linear regression and mixed effects models where chemokine level was the outcome and whole blood RNA transcript levels for CXCL9 and CXCL10, blood immune cell populations, or different classifications of immunosuppression use (see Supplementary Materials) were the predictors of interest. We calculated correlation coefficients (r values) for these models by taking the square-root of the adjusted R2 from the linear regression equations; for mixed effects linear regression models, the R2 values were initially calculated using methods as described by Snijders and Bosker [
      • Snijders T.A.B.
      • Bosker R.J.
      Modeled variance in two-level models.
      ]. Where indicated, we adjusted regression models for several confounders including age, sex, race, binary designations for immunosuppression use (yes/no), and prior smoking history (yes/no). All statistical analyses were done using Stata/SE 15.1 software (StataCorp LLC, College Station, TX) and GraphPad Prism 6 software (GraphPad Software, Inc., La Jolla, CA) was used to construct figures.

      3. Results

      3.1 Characteristics of sarcoidosis subjects

      One hundred and eight sarcoidosis subjects had available longitudinal blood and clinical measurements for this analysis. There were 103/108 subjects who had samples available for chemokine measurements from the initial study visit; the remaining 5/108 had measurements at the second and/or later visits (Table 1). Forty-nine percent of subjects were taking systemic immunosuppressive therapy at the initial study visit and the majority of subjects (74%) had extra-thoracic involvement defined by physician assessment of medical records (Table 2).
      Table 1Demographics at each visit.
      All SubjectsInitial Visit6-month follow-up12-month follow-up24-month follow-up
      Total number per visit108103675936
      Age (years ± SD)50 ± 1051 ± 1052 ± 1153 ± 1156 ± 11
      Female (%)67 (62)64 (62)37 (54)32 (54)19 (53)
      Race (%)
       African American15 (14)15 (15)7 (10)5 (8)5 (14)
       White75 (69)71 (69)48 (71)45 (76)25 (69)
       Hispanic8 (7.4)8 (7.8)7 (10)5 (8)4 (11)
       Other Ethnicity10 (9.3)9 (8.7)6 (9)4 (7)2 (6)
      Ever Smokers (%)50 (46)47 (46)30 (44)23 (39)14 (39)
      Table 2Clinical characteristics of sarcoidosis subjects at their initial visit.
      Imaging: Scadding StageN (%)
      011
      Two subjects with Scadding stage 0 had prior lung involvement, the other subjects either had neurologic or skin involvement.
      (10)
      113 (12)
      250 (46)
      310 (9)
      424 (22)
      Immunosuppression use49 (45)
      Extra-thoracic involvement74 (69)
      Pulmonary Function Tests
      NMean (SD)Range
      FVC %predicted10096 (15)59–140
      FEV1 %predicted10090 (18)28–140
      FEV1/FVC1000.75 (0.088)0.34–0.95
      DLCO %predicted7371 (14)39–108
      TLC %predicted6896 (15)59–131
      Abbreviations: DLCO = Diffusing Capacity of the Lungs for Carbon Monoxide, FEV1 = Forced Expiratory Volume in 1 Second, FEV1/FVC = Forced Expiratory Volume in 1 Second to Forced Vital Capacity ratio, FVC = Forced Vital Capacity, TLC = Total Lung Capacity.
      a Two subjects with Scadding stage 0 had prior lung involvement, the other subjects either had neurologic or skin involvement.

      3.1.1 CXCL10 had greater correlation with pulmonary function and respiratory symptoms relative to CXCL9

      We previously found that CXCL9 and CXCL10 levels were not perfectly correlated with each other at study enrollment (Spearman r = 0.72) [
      • Arger N.K.
      • Ho M.
      • Woodruff P.G.
      • Koth L.L.
      Serum CXCL11 correlates with pulmonary outcomes and disease burden in sarcoidosis.
      ]. Thus, we hypothesized that these chemokines may relate differently to specific outcomes, including lung function. We first correlated lung function with serum levels of each chemokine at initial subject visit while adjusting for prior smoking and immunosuppression use (Fig. 1). We found that %predicted FVC was 17% points lower (~670 mL in this cohort) for each 10-fold increase in CXCL10 (e.g. 1.6 to 2.6 log10 units on the log scale) (Fig. 1A). We found similar results for FEV1, DLCO, and TLC (Fig. 1B–D, Supplementary Table S1).
      Fig. 1
      Fig. 1Correlations between pulmonary function measurements and serum CXCL10 levels at initial subject visit. A) FVC, B) FEV1, C) DLCO, and D) TLC. Data are displayed as log10 transformations of serum CXCL10 levels (individual values denoted by open black circles) and fitted lines for predicted pulmonary function values (dashed lines). The β-coefficients show how the average %predicted pulmonary function values vary for every log10-increase in CXCL10 (see ) (*p < 0.05, **P < 0.01). Both fitted lines and β-coefficients were adjusted for prior smoking and immunosuppression use. Abbreviations: FEV1 = Forced Expiratory Volume in 1 Second, FVC= Forced Vital Capacity, DLCO = Diffusing Capacity for Carbon Monoxide.
      Our primary analysis goal was to examine the relationships between repeated measurements of lung function and chemokine levels over the initial two-year follow-up period. We used mixed effects models and adjusted for prior smoking and immunosuppression use. Higher levels of CXCL10 were associated with lower FVC, TLC and DLCO values (Table 3). This indicates that in a given individual, the average %predicted FVC was lower by 4.7% points for every 10-fold increase in CXCL10 at any visit within the first two years of the study. In contrast, CXCL9 was only correlated with DLCO (Table 3 and Supplementary Table S1). Of note, the total ranges for log10(CXCL10) and log10(CXCL9) in this study were 0.84–2.7 and 1.3–3.3, respectively.
      Table 3Results from mixed effects regression models of respiratory variables measured repeatedly over two years.
      OutcomeMain Predictor
      Chemokine variables analyzed as log10 transformations; models adjusted for immunosuppression use and prior smoking.
      β-coefficient95% CIp-value
      FVC %predCXCL91.1(-2.5, 4.8)0.54
      CXCL10−4.6(-9.1, 0.68)0.047
      FEV1 %predCXCL91.9(-1.6, 5.4)0.28
      CXCL10−1.6(-6.0, 2.8)0.48
      DLCO %predCXCL9−12(-19, 5.4)4.5x10−4
      CXCL10−15(-24, 6.7)5.0x10−4
      TLC %predCXCL9−2.2(-7.9, 3.6)0.46
      CXCL10−9.5(-17, −2.5)0.0076
      Dyspnea ScoreCXCL917%
      Coefficients for dyspnea score are shown as the % change in score for each 10-fold increase in chemokine level.
      (15%, 61%)0.34
      CXCL1058%(5.0%, 140%)0.028
      Abbreviations: CI = Confidence Interval, DLCO = Diffusing Capacity of the Lungs for Carbon Monoxide, FEV1 = Forced Expiratory Volume in 1 Second, FVC = Forced Vital Capacity, %pred = % predicted, TLC = Total Lung Capacity.
      a Chemokine variables analyzed as log10 transformations; models adjusted for immunosuppression use and prior smoking.
      b Coefficients for dyspnea score are shown as the % change in score for each 10-fold increase in chemokine level.
      To further assess pulmonary involvement by chest radiography obtained at initial subject visit, we compared chemokine levels by Scadding stage [
      • Benn B.S.
      • Lehman Z.
      • Kidd S.A.
      • Ho M.
      • Sun S.
      • Ramstein J.
      • Arger N.K.
      • Nguyen C.P.
      • Su R.
      • Gomez A.
      • Gelfand J.M.
      • Koth L.L.
      Clinical and biological insights from the university of California san Francisco prospective and longitudinal cohort.
      ]. In analyses that compared chemokines between Scadding stages 1 through 4, there were no differences in chemokine levels between stages in either unadjusted (ANOVA) or adjusted (linear regression) models. Also, neither of the chemokines differed between subjects with or without fibrosis on chest radiography.
      We also assessed whether CXCL10 and CXCL9 correlated longitudinally with respiratory symptoms as assessed by the UCSD Dyspnea score, where a higher score indicates more dyspnea [
      • Eakin E.G.
      • Resnikoff P.M.
      • Prewitt L.M.
      • Ries A.L.
      • Kaplan R.M.
      Validation of a new dyspnea measure: the UCSD shortness of breath questionnaire.
      ,
      • Swigris J.J.
      • Yorke J.
      • Sprunger D.B.
      • Swearingen C.
      • Pincus T.
      • du Bois R.M.
      • Brown K.K.
      • Fischer A.
      Assessing dyspnea and its impact on patients with connective tissue disease-related interstitial lung disease.
      ,
      • Swigris J.J.
      • Han M.
      • Vij R.
      • Noth I.
      • Eisenstein E.L.
      • Anstrom K.J.
      • Brown K.K.
      • Fairclough D.
      The UCSD shortness of breath questionnaire has longitudinal construct validity in idiopathic pulmonary fibrosis.
      ]. Using a similar mixed effects model as for the pulmonary function measurements, we found that for every 10-fold increase in CXCL10, the average shortness of breath score increased by 58% (Table 3). To investigate whether this effect on symptom severity was mediated by lower pulmonary function, we also included a model with % predicted FVC and DLCO. After this adjustment, CXCL10 was still statistically significantly correlated with shortness of breath score (122% increase for every 10-fold increase in CXCL10, 95% CI 6.4%–370%, p = 0.038). The correlation between longitudinal dyspnea scores and CXCL9 was lower and not statistically significant in similar analyses (Table 3).

      3.1.2 CXCL10 had greater predictive value for pulmonary function declines

      To assess whether chemokine levels measured during the first two years of the study were associated with pulmonary function declines at any point in the ~5 years (66 months) of total follow-up, we performed a time to event analysis. First, we identified subjects with a decline in absolute FVC or DLCO values of ≥10% or ≥15%, respectively [
      • Keir G.
      • Wells A.U.
      Assessing pulmonary disease and response to therapy: which test?.
      ,
      • Baughman R.P.
      • Teirstein A.S.
      • Judson M.A.
      • Rossman M.D.
      • Yeager Jr., H.
      • Bresnitz E.A.
      • DePalo L.
      • Hunninghake G.
      • Iannuzzi M.C.
      • Johns C.J.
      • McLennan G.
      • Moller D.R.
      • Newman L.S.
      • Rabin D.L.
      • Rose C.
      • Rybicki B.
      • Weinberger S.E.
      • Terrin M.L.
      • Knatterud G.L.
      • Cherniak R.
      Clinical characteristics of patients in a case control study of sarcoidosis.
      ], at any time after subjects’ initial visit. Next, we performed analyses to determine if the risk of pulmonary function decline was increased with higher chemokine levels (represented as either binary variables designating those above or below the median chemokine value or continuous chemokine level variables). In unadjusted analysis, subjects with a CXCL10 level above the median (176 pg/mL) had a higher risk of eventual decline (log-rank p = 0.037). In the Cox proportional hazards models, we included age, sex, race, and prior smoking as co-variates, but we stratified by immunosuppression use since this variable violated the proportional hazards assumption based on the Schoenfeld test [
      • Schoenfeld D.
      Partial residuals for the proportional hazards regression model.
      ]. Subjects with CXCL10 levels above the median had 4.1 times the risk of pulmonary function decline (HR = 4.1, 95% CI 1.5–12, p = 0.0078) (Fig. 2A). Modeled as a continuous variable, we found that each 10-fold increase in CXCL10 was associated with 8.8 times the risk of experiencing a decline (HR for log10(CXCL10) = 8.8, 95% CI 1.6–50, p = 0.014). This relationship did not meet statistical significance for CXCL9 (log rank p = 0.40, adjusted HR = 1.9 with p = 0.17 for CXCL9 dichotomized at the median (119 pg/mL); adjusted HR for log10(CXCL9) = 2.2, p = 0.15) (Fig. 2B). As a sensitivity analysis to address subjects censored prior to complete follow-up, we tested different assumptions about event rates in those censored. Whether we assumed all censored subjects made it to the five-year follow-up without a decline, all censored subjects had decline at the time of censoring, or the same proportion of randomly chosen censored subjects had a decline at the time of censoring as those who made it to five-year follow-up, the direction and magnitude of the effects of our predictors on the hazard ratios or log-rank statistics as reported above did not change.
      Fig. 2
      Fig. 2Relationship between serum CXCL10 and CXCL9 and longitudinal decline in lung function.
      We defined lung function decline as a percent change in absolute forced vital capacity or diffusing capacity of 10% or 15%, respectively, over the study period (~
      5 years). Total time at risk was 291 person-years. We used Cox proportional hazards models adjusted for age, race, sex, prior smoking, and immunosuppression use and dichotomized subjects based on chemokine levels above or below median values to assess the predictive value of these chemokines for lung function decline. Abbreviations: HR =
      hazard ratio.

      3.1.3 CXCL9, but not CXCL10, positively correlated with organ involvement

      Our second main analytic goal was to assess relationships between total systemic organ involvement and levels of CXCL9 and CXCL10 at subjects’ initial visit [
      • Arger N.K.
      • Ho M.
      • Woodruff P.G.
      • Koth L.L.
      Serum CXCL11 correlates with pulmonary outcomes and disease burden in sarcoidosis.
      ]. First, we compared chemokine levels in subjects with one versus greater than one organ involved, and then determined if total organ number increased incrementally with higher levels of each chemokine. In unadjusted analyses, we found higher CXCL9 levels in subjects who had greater than one organ involved compared to only one organ (mean CXCL9 ± SD was 258 ± 360 versus 123 ± 71 pg/mL, p = 0.034, N = 37 vs. 64, respectively) (Fig. 3A). After adjusting for age, sex, race, and immunosuppression use, we found that for every 10-fold increase in CXCL9, the odds of having more than one organ involved increased 5.7 times (odds ratio (OR) = 5.7, p = 0.017, 95% CI 1.4–24). Next, using a Poisson regression adjusted for age, sex, race, and immunosuppression, we found a statistically significant increase in organ number with higher CXCL9 levels (log10(CXCL9) β = 1.5, 95% CI 1.1–2.0, p = 0.022), indicating that for every 10-fold increase in CXCL9, between 1 and 2 additional organs were involved (Fig. 3B). In contrast, there were no differences in CXCL10 levels between those with one versus greater than one organ involved in unadjusted (p = 0.13) or adjusted (OR 95% CI 0.41–9.4, p = 0.43) analyses and there was no statistically significant correlation between organ involvement and CXCL10 using Poisson regression (95% CI 0.74–1.9, p = 0.47).
      Fig. 3
      Fig. 3Relationship between serum CXCL9 levels and the number of organs involved with sarcoidosis.
      Organ assessments were performed by study physician review of the patient's records at initial subject visit. A) Subjects categorized as having one or more than one organ involved. B) Relationship between total organ involvement and CXCL9. Data are displayed as log10 transformations of CXCL9 levels (individual values denoted by open black circles). The lower limits of assay detection denoted by the dashed line in A). The dashed line in B) represents the fitted line for organ number adjusted for age, sex, race, and immunosuppression use.
      Finally, we used logistic regression to assess if either chemokine level was higher in subjects with specific organ system involvement. In these analyses, we required 10 or more subjects to have that organ involved to provide a sufficient sample size. Higher CXCL9 levels increased the odds of extra-thoracic lymph node involvement (OR log10(CXCL9) = 6.7, 95% CI 1.6–28, p = 0.0090) and ocular disease (OR log10(CXCL9) = 12, 95% CI 2.3–58, p = 0.0029). In contrast, CXCL10 levels were not significantly associated with either extra-thoracic lymph node involvement (OR 95% CI 0.30–9.2, p = 0.57) or ocular involvement (OR 95% CI 0.74–150, p = 0.083).

      3.1.4 CXCL10, not CXCL9, correlated with its own gene expression in peripheral blood and blood monocyte counts

      Because we found that CXCL9 and CXCL10 were predictive of different clinical sarcoidosis outcomes, we wanted to explore potential reasons for these differences, specifically if there was evidence that the two chemokines were differentially produced by cells in the blood. Given that several blood immune cell populations can produce these chemokines, including monocytes and T cells [
      • Luster A.D.
      • Ravetch J.V.
      Biochemical characterization of a gamma interferon-inducible cytokine (IP-10).
      ,
      • Zipfel P.F.
      • Bialonski A.
      • Skerka C.
      Induction of members of the IL-8/NAP-1 gene family in human T lymphocytes is suppressed by cyclosporin A.
      ,
      • Padovan E.
      • Spagnoli G.C.
      • Ferrantini M.
      • Heberer M.
      IFN-alpha2a induces IP-10/CXCL10 and MIG/CXCL9 production in monocyte-derived dendritic cells and enhances their capacity to attract and stimulate CD8+ effector T cells.
      ], we analyzed gene expression data previously generated from the same blood samples used to measure the chemokine proteins [
      • Su R.
      • Li M.M.
      • Bhakta N.R.
      • Solberg O.D.
      • Darnell E.P.
      • Ramstein J.
      • Garudadri S.
      • Ho M.
      • Woodruff P.G.
      • Koth L.L.
      Longitudinal analysis of sarcoidosis blood transcriptomic signatures and disease outcomes.
      ]. In cross-sectional and longitudinal mixed effects linear regression analyses adjusted for age, sex, race, and immunosuppression use, we found that CXCL10 protein level was positively correlated with CXCL10 expression, but the degree of correlation between CXCL9 expression and CXCL9 protein was lower and did not meet statistical significance (Table 4 and Fig. 4). For every doubling of CXCL10 expression, CXCL10 protein level increases by 32% (p = 1.0 × 10−6), suggesting that peripheral blood could be a significant source of CXCL10, whereas the main source of CXCL9 may be from other tissues.
      Table 4Results from regression models for CXCL9 or CXCL10 levels with either their respective mRNA transcript levels or peripheral blood monocyte concentrations.
      Cross-sectional Models at Initial Visit
      Linear regression models with measurements obtained at initial subject visits and adjusted for age, race, sex, and immunosuppression use.
      OutcomeMain Predictor
      Corresponding CXCL9 or CXCL10 Level (Log2 [Relative Expression])
      CXCL9 expression range: −1.9 – 5.6; CXCL10 expression range −2.8 – 3.0.
      β-coefficient
      Non-linear combinations of β-coefficients performed to show the %change in chemokine level for every unit increase in predictor.
      95% CIp-valuer value
      CXCL916%(6.5, 43)0.180.15
      CXCL1032%(19, 47)1.0x10−60.54
      Longitudinal Models
      Mixed effects models with measurements obtained over two years and adjusted for age, race, sex, and immunosuppression use.
      OutcomeMain Predictor
      Corresponding CXCL9 or CXCL10 Level (Log2[Relative Expression])
      β-coefficient95% CIp-valuer value
      CXCL96.7%(-0.30, 13)0.0580.31
      CXCL1016%(9.2, 24)2.9x10−60.54
      Monocytes (106 cells/mL)
      Monocytes concentrations had a total range of 1.5 × 106 cells/mL.
      β-coefficient95% CIp-valuer value
      CXCL9−3.7%(-35, 44)0.860.29
      CXCL1047%(5.6, 110)0.0220.43
      Abbreviations: CI = Confidence Interval.
      a Linear regression models with measurements obtained at initial subject visits and adjusted for age, race, sex, and immunosuppression use.
      b CXCL9 expression range: −1.9 – 5.6; CXCL10 expression range −2.8 – 3.0.
      c Non-linear combinations of β-coefficients performed to show the %change in chemokine level for every unit increase in predictor.
      d Mixed effects models with measurements obtained over two years and adjusted for age, race, sex, and immunosuppression use.
      e Monocytes concentrations had a total range of 1.5 × 106 cells/mL.
      Fig. 4
      Fig. 4Relationship between serum chemokines and their respective whole blood mRNA gene transcript level at initial subject visit.
      We analyzed paired chemokine and mRNA gene transcript levels measured from the same blood sample and obtained at subjects' initial visit using linear regression analysis. Data for A) CXCL10 and B) CXCL9 are displayed as log10 transformations of chemokine values and log2 transformations of relative gene expression values (individual values denoted by open black circles) with the fitted lines for the chemokines adjusted for age, sex, race, and immunosuppression. The β-coefficients show the % increase in chemokine level for every log2-unit increase in gene expression adjusted for age, sex, race, and immunosuppression use (See ); CXCL10 **p = 1.0 × 10−6, CXCL9 p = 0.18.
      To identify a potential immune cellular source of CXCL10 in the blood, we assessed the relationships between CXCL10 chemokine levels and concentrations of blood immune cell populations as measured by clinical laboratory testing. In analyses using repeated measures of the blood markers, we found that the counts of peripheral blood monocytes (but not white blood cells, lymphocytes, or neutrophils) were positively associated with serum CXCL10 level suggesting that monocytes could be a source of CXCL10 in the blood (Table 4). Congruent with the correlations of CXCL9 protein to CXCL9 gene expression, we found no relationship between serum levels of CXCL9 and blood monocytes.

      3.1.5 CXCL9 and CXCL10 are both negatively correlated with immunosuppression use

      Because immunosuppression use could influence CXCL9 and CXCL10 levels through reducing inflammation, we controlled for immunosuppression as part of our analysis plan using a binary variable (any immunosuppression use or none at a given visit). However, we also wanted to determine how immunosuppression itself influenced chemokine levels. Using the observational data collected from this cohort on the types and amounts of immunosuppression at each blood draw, we performed separate mixed effects models where either CXCL9 or CXCL10 was the dependent variable and immunosuppression use was the predictor (using binary or continuous metrics). Controlling for age, sex, and race in these models, we found that CXCL9 and CXCL10 levels were 28–29% lower in subjects taking any immunosuppression at a given blood draw (Table 5). Using a different model that included two separate terms for immunosuppression use with prednisone dose in mg/day as a continuous variable and DMARD use (e.g. methotrexate or azathioprine) as present or not, we found that higher prednisone levels resulted in lower CXCL10 levels and DMARD use was associated with lower levels of both chemokines (Table 5).
      Table 5Results from mixed effects regression analysis of CXCL9 or CXCL10 obtained over two years and immunosuppression use at time of measurement modeled in two different ways.
      OutcomeMain Predictor(s)
      Models adjusted for age, race, and sex.
      Model 1: Binary Immunosuppression Use (Yes/No)
      β-coefficient
      Non-linear combinations of β-coefficients performed to show the %change in chemokine level for every unit increase in continuous predictor or if binary variable = Yes.
      (95% CI)p-value
      CXCL9−29%(-42, −14)4.9x10−4
      CXCL10−28%(-37, −17)6.4x10−6
      Model 2: Prednisone Dose (mg/day)
      Prednisone dose in units of 1 mg/day.
      DMARD Use (Yes/No)
      β-coefficient(95% CI)p-valueβ coefficient(95% CI)p-value
      CXCL9−0.70%(-1.6, 0.17)0.12−35%(-47, −20)5.0x10−5
      CXCL10−9.6%(-1.7, −0.33)0.0034−22%(-34, −11)5.1x10−4
      Abbreviations: CI = Confidence Interval, DMARD = disease-modifying antirheumatic drug.
      a Models adjusted for age, race, and sex.
      b Non-linear combinations of β-coefficients performed to show the %change in chemokine level for every unit increase in continuous predictor or if binary variable = Yes.
      c Prednisone dose in units of 1 mg/day.

      4. Discussion

      Sarcoidosis is a systemic disease involving granulomatous inflammation with upregulation of immune pathways related to IFN-γ [
      • Mollers M.
      • Aries S.P.
      • Dromann D.
      • Mascher B.
      • Braun J.
      • Dalhoff K.
      Intracellular cytokine repertoire in different T cell subsets from patients with sarcoidosis.
      ,
      • Wahlstrom J.
      • Katchar K.
      • Wigzell H.
      • Olerup O.
      • Eklund A.
      • Grunewald J.
      Analysis of intracellular cytokines in CD4+ and CD8+ lung and blood T cells in sarcoidosis.
      ,
      • Koth L.L.
      • Solberg O.D.
      • Peng J.C.
      • Bhakta N.R.
      • Nguyen C.P.
      • Woodruff P.G.
      Sarcoidosis blood transcriptome reflects lung inflammation and overlaps with tuberculosis.
      ,
      • Prior C.
      • Haslam P.L.
      Increased levels of serum interferon-gamma in pulmonary sarcoidosis and relationship with response to corticosteroid therapy.
      ]. In this study, our goals were to compare serum levels of two interferon-induced chemokines, CXCL9 and CXCL10, with respect to important clinical outcomes in sarcoidosis. Because in vivo and in vitro observations have shown differences in the types of inflammatory stimuli that can induce these chemokines [
      • Groom J.R.
      • Luster A.D.
      CXCR3 ligands: redundant, collaborative and antagonistic functions.
      ,
      • Wang Q.
      • Nagarkar D.R.
      • Bowman E.R.
      • Schneider D.
      • Gosangi B.
      • Lei J.
      • Zhao Y.
      • McHenry C.L.
      • Burgens R.V.
      • Miller D.J.
      • Sajjan U.
      • Hershenson M.B.
      Role of double-stranded RNA pattern recognition receptors in rhinovirus-induced airway epithelial cell responses.
      ,
      • Amichay D.
      • Gazzinelli R.T.
      • Karupiah G.
      • Moench T.R.
      • Sher A.
      • Farber J.M.
      Genes for chemokines MuMig and Crg-2 are induced in protozoan and viral infections in response to IFN-gamma with patterns of tissue expression that suggest nonredundant roles in vivo.
      ,
      • Nakanishi Y.
      • Lu B.
      • Gerard C.
      • Iwasaki A.
      CD8(+) T lymphocyte mobilization to virus-infected tissue requires CD4(+) T-cell help.
      ,
      • Medoff B.D.
      • Wain J.C.
      • Seung E.
      • Jackobek R.
      • Means T.K.
      • Ginns L.C.
      • Farber J.M.
      • Luster A.D.
      CXCR3 and its ligands in a murine model of obliterative bronchiolitis: regulation and function.
      ,
      • Ohmori Y.
      • Wyner L.
      • Narumi S.
      • Armstrong D.
      • Stoler M.
      • Hamilton T.A.
      Tumor necrosis factor-alpha induces cell type and tissue-specific expression of chemoattractant cytokines in vivo.
      ,
      • Ciesielski C.J.
      • Andreakos E.
      • Foxwell B.M.
      • Feldmann M.
      TNFalpha-induced macrophage chemokine secretion is more dependent on NF-kappaB expression than lipopolysaccharides-induced macrophage chemokine secretion.
      ,
      • Luster A.D.
      • Unkeless J.C.
      • Ravetch J.V.
      Gamma-interferon transcriptionally regulates an early-response gene containing homology to platelet proteins.
      ,
      • Proost P.
      • Verpoest S.
      • Van de Borne K.
      • Schutyser E.
      • Struyf S.
      • Put W.
      • Ronsse I.
      • Grillet B.
      • Opdenakker G.
      • Van Damme J.
      Synergistic induction of CXCL9 and CXCL11 by Toll-like receptor ligands and interferon-gamma in fibroblasts correlates with elevated levels of CXCR3 ligands in septic arthritis synovial fluids.
      ,
      • Proost P.
      • Vynckier A.K.
      • Mahieu F.
      • Put W.
      • Grillet B.
      • Struyf S.
      • Wuyts A.
      • Opdenakker G.
      • Van Damme J.
      Microbial Toll-like receptor ligands differentially regulate CXCL10/IP-10 expression in fibroblasts and mononuclear leukocytes in synergy with IFN-gamma and provide a mechanism for enhanced synovial chemokine levels in septic arthritis.
      ], we were interested in assessing whether the circulating levels of each chemokine were differentially associated with specific clinical outcomes. For CXCL10, we found negative correlations with lung function measurements both at entry into the cohort and over time and we found that higher CXCL10 levels during the first two years of follow up increased the risk of having a future clinically significant decline in FVC or DLCO during the 5-year study period. Higher CXCL0 levels also correlated with greater dyspnea scores in longitudinal analyses. There was less association of CXCL9 with these same outcomes. In contrast, when examining the endpoint of organ involvement, we found that CXCL9 was positively associated with the total number of organs involved as well as for specific organs, such as ocular or extra-thoracic lymph node involvement, while CXCL10 was not. In a prior study, we examined the relationship of these same outcomes with levels of CXCL11 [
      • Arger N.K.
      • Ho M.
      • Woodruff P.G.
      • Koth L.L.
      Serum CXCL11 correlates with pulmonary outcomes and disease burden in sarcoidosis.
      ]. Interestingly, we found that higher CXCL11 levels at enrollment increased the risk of future DLCO and FVC decline and positively correlated with number of organs involved. We speculate that a reason for these different associations between the three chemokines and clinical outcomes may be related to differences in receptor recognition and cellular sources of production [
      • Groom J.R.
      • Luster A.D.
      CXCR3 ligands: redundant, collaborative and antagonistic functions.
      ,
      • Hancock W.W.
      • Gao W.
      • Csizmadia V.
      • Faia K.L.
      • Shemmeri N.
      • Luster A.D.
      Donor-derived IP-10 initiates development of acute allograft rejection.
      ,
      • Burns J.M.
      • Summers B.C.
      • Wang Y.
      • Melikian A.
      • Berahovich R.
      • Miao Z.
      • Penfold M.E.
      • Sunshine M.J.
      • Littman D.R.
      • Kuo C.J.
      • Wei K.
      • McMaster B.E.
      • Wright K.
      • Howard M.C.
      • Schall T.J.
      A novel chemokine receptor for SDF-1 and I-TAC involved in cell survival, cell adhesion, and tumor development.
      ,
      • Zohar Y.
      • Wildbaum G.
      • Novak R.
      • Salzman A.L.
      • Thelen M.
      • Alon R.
      • Barsheshet Y.
      • Karp C.L.
      • Karin N.
      CXCL11-dependent induction of FOXP3-negative regulatory T cells suppresses autoimmune encephalomyelitis.
      ,
      • Colvin R.A.
      • Campanella G.S.
      • Sun J.
      • Luster A.D.
      Intracellular domains of CXCR3 that mediate CXCL9, CXCL10, and CXCL11 function.
      ,
      • Cole K.E.
      • Strick C.A.
      • Paradis T.J.
      • Ogborne K.T.
      • Loetscher M.
      • Gladue R.P.
      • Lin W.
      • Boyd J.G.
      • Moser B.
      • Wood D.E.
      • Sahagan B.G.
      • Neote K.
      Interferon-inducible T cell alpha chemoattractant (I-TAC): a novel non-ELR CXC chemokine with potent activity on activated T cells through selective high affinity binding to CXCR3.
      ]. Taken together, the findings suggest that each chemokine could be used to help predict specific clinical outcomes in sarcoidosis patients.
      Our findings are consistent with prior sarcoidosis studies that found elevated levels of CXCL9 and/or CXCL10 in the lung (lavage fluid, lavage cells, or tissue) [
      • Agostini C.
      • Cassatella M.
      • Zambello R.
      • Trentin L.
      • Gasperini S.
      • Perin A.
      • Piazza F.
      • Siviero M.
      • Facco M.
      • Dziejman M.
      • Chilosi M.
      • Qin S.
      • Luster A.D.
      • Semenzato G.
      Involvement of the IP-10 chemokine in sarcoid granulomatous reactions.
      ,
      • Miotto D.
      • Christodoulopoulos P.
      • Olivenstein R.
      • Taha R.
      • Cameron L.
      • Tsicopoulos A.
      • Tonnel A.B.
      • Fahy O.
      • Lafitte J.J.
      • Luster A.D.
      • Wallaert B.
      • Mapp C.E.
      • Hamid Q.
      Expression of IFN-gamma-inducible protein; monocyte chemotactic proteins 1, 3, and 4; and eotaxin in TH1- and TH2-mediated lung diseases.
      ,
      • Cui A.
      • Anhenn O.
      • Theegarten D.
      • Ohshimo S.
      • Bonella F.
      • Sixt S.U.
      • Peters J.
      • Sarria R.
      • Guzman J.
      • Costabel U.
      Angiogenic and angiostatic chemokines in idiopathic pulmonary fibrosis and granulomatous lung disease.
      ,
      • Arakelyan A.
      • Kriegova E.
      • Kubistova Z.
      • Mrazek F.
      • Kverka M.
      • du Bois R.M.
      • Kolek V.
      • Petrek M.
      Protein levels of CC chemokine ligand (CCL)15, CCL16 and macrophage stimulating protein in patients with sarcoidosis.
      ,
      • Piotrowski W.J.
      • Mlynarski W.
      • Fendler W.
      • Wyka K.
      • Marczak J.
      • Gorski P.
      • Antczak A.
      Chemokine receptor CXCR3 ligands in bronchoalveolar lavage fluid: associations with radiological pattern, clinical course, and prognosis in sarcoidosis.
      ,
      • Li H.
      • Zhao X.
      • Wang J.
      • Zong M.
      • Yang H.
      Bioinformatics analysis of gene expression profile data to screen key genes involved in pulmonary sarcoidosis.
      ] or the lung and blood [
      • Antoniou K.M.
      • Tzouvelekis A.
      • Alexandrakis M.G.
      • Sfiridaki K.
      • Tsiligianni I.
      • Rachiotis G.
      • Tzanakis N.
      • Bouros D.
      • Milic-Emili J.
      • Siafakas N.M.
      Different angiogenic activity in pulmonary sarcoidosis and idiopathic pulmonary fibrosis.
      ,
      • Sugiyama K.
      • Mukae H.
      • Ishii H.
      • Kakugawa T.
      • Ishimoto H.
      • Nakayama S.
      • Shirai R.
      • Fujii T.
      • Mizuta Y.
      • Kohno S.
      Elevated levels of interferon gamma-inducible protein-10 and epithelial neutrophil-activating peptide-78 in patients with pulmonary sarcoidosis.
      ,
      • Nishioka Y.
      • Manabe K.
      • Kishi J.
      • Wang W.
      • Inayama M.
      • Azuma M.
      • Sone S.
      CXCL9 and 11 in patients with pulmonary sarcoidosis: a role of alveolar macrophages.
      ,
      • Nureki S.
      • Miyazaki E.
      • Ando M.
      • Ueno T.
      • Fukami T.
      • Kumamoto T.
      • Sugisaki K.
      • Tsuda T.
      Circulating levels of both Th1 and Th2 chemokines are elevated in patients with sarcoidosis.
      ,
      • Pignatti P.
      • Brunetti G.
      • Moretto D.
      • Yacoub M.R.
      • Fiori M.
      • Balbi B.
      • Balestrino A.
      • Cervio G.
      • Nava S.
      • Moscato G.
      Role of the chemokine receptors CXCR3 and CCR4 in human pulmonary fibrosis.
      ]. Most of these studies found higher chemokine levels when comparing all sarcoidosis subjects to healthy controls. Some studies compared Scadding stage I or II subjects to healthy controls [
      • Arakelyan A.
      • Kriegova E.
      • Kubistova Z.
      • Mrazek F.
      • Kverka M.
      • du Bois R.M.
      • Kolek V.
      • Petrek M.
      Protein levels of CC chemokine ligand (CCL)15, CCL16 and macrophage stimulating protein in patients with sarcoidosis.
      ,
      • Piotrowski W.J.
      • Mlynarski W.
      • Fendler W.
      • Wyka K.
      • Marczak J.
      • Gorski P.
      • Antczak A.
      Chemokine receptor CXCR3 ligands in bronchoalveolar lavage fluid: associations with radiological pattern, clinical course, and prognosis in sarcoidosis.
      ,
      • Busuttil A.
      • Weigt S.S.
      • Keane M.P.
      • Xue Y.Y.
      • Palchevskiy V.
      • Burdick M.D.
      • Huang C.
      • Zisman D.A.
      • Fishbein M.
      • Lynch 3rd, J.P.
      • Strieter R.M.
      • Elashoff R.M.
      • Belperio J.A.
      CXCR3 ligands are augmented during the pathogenesis of pulmonary sarcoidosis.
      ,
      • Schnerch J.
      • Prasse A.
      • Vlachakis D.
      • Schuchardt K.L.
      • Pechkovsky D.V.
      • Goldmann T.
      • Gaede K.I.
      • Muller-Quernheim J.
      • Zissel G.
      Functional toll-like receptor 9 expression and CXCR3 ligand release in pulmonary sarcoidosis.
      ]. Two studies found lower levels of these chemokines in subjects with Löfgren's syndrome relative non-Löfgren sarcoidosis [
      • Arakelyan A.
      • Kriegova E.
      • Kubistova Z.
      • Mrazek F.
      • Kverka M.
      • du Bois R.M.
      • Kolek V.
      • Petrek M.
      Protein levels of CC chemokine ligand (CCL)15, CCL16 and macrophage stimulating protein in patients with sarcoidosis.
      ,
      • Piotrowski W.J.
      • Mlynarski W.
      • Fendler W.
      • Wyka K.
      • Marczak J.
      • Gorski P.
      • Antczak A.
      Chemokine receptor CXCR3 ligands in bronchoalveolar lavage fluid: associations with radiological pattern, clinical course, and prognosis in sarcoidosis.
      ]. These studies were cross-sectional in design, except one that measured lung lavage chemokine levels and did not find these levels to be predictive of radiographic remission at two-year follow-up [
      • Piotrowski W.J.
      • Mlynarski W.
      • Fendler W.
      • Wyka K.
      • Marczak J.
      • Gorski P.
      • Antczak A.
      Chemokine receptor CXCR3 ligands in bronchoalveolar lavage fluid: associations with radiological pattern, clinical course, and prognosis in sarcoidosis.
      ]. The strength of our study was the fact that it included a longitudinal study design to correlate pulmonary physiology and chemokine levels, with both values measured repeatedly. We also carried out detailed organ phenotyping amongst sarcoidosis subjects, allowing us to assess relationships between chemokine levels and organ involvement at entry into the cohort.
      We also observed that higher immunosuppression usage was associated with lower chemokine levels. Our finding that DMARD use was associated with lower levels of both chemokines, whereas only CXCL10 levels were lower with higher doses of prednisone could potentially be due to differing effects of prednisone on the sources of CXCL10 and CXCL9. However, given that these were correlative analyses, there could be other non-causal explanations. We acknowledge that our study was not a randomized clinical trial and was not designed to differentiate patients with active versus resolved sarcoidosis or assess the effect of treatment on these protein levels, however our findings suggest that these serum chemokine levels have potential as prognostic markers for both pulmonary outcomes and response to therapy. There is precedent for using CXCL9 and CXCL10 as markers of disease activity in other granulomatous diseases. CXCL9 and CXCL10 levels have been found to be predictive of disease progression and response to therapy in tuberculosis [
      • Kim S.
      • Lee H.
      • Kim H.
      • Kim Y.
      • Cho J.E.
      • Jin H.
      • Kim D.Y.
      • Ha S.J.
      • Kang Y.A.
      • Cho S.N.
      • Lee H.
      Diagnostic performance of a cytokine and IFN-gamma-induced chemokine mRNA assay after Mycobacterium tuberculosis-specific antigen stimulation in whole blood from infected individuals.
      ,
      • Lee K.
      • Chung W.
      • Jung Y.
      • Kim Y.
      • Park J.
      • Sheen S.
      • Park K.
      CXCR3 ligands as clinical markers for pulmonary tuberculosis.
      ,
      • Chung W.
      • Lee K.
      • Jung Y.
      • Kim Y.
      • Park J.
      • Sheen S.
      • Lee J.
      • Kang D.
      • Park K.
      Serum CXCR3 ligands as biomarkers for the diagnosis and treatment monitoring of tuberculosis.
      ]. Chung et al., showed that protein levels of CXCL9 and CXCL10 were increased in those with confirmed tuberculosis infection compared to those without active infection and both protein levels decreased after successful treatment [
      • Chung W.
      • Lee K.
      • Jung Y.
      • Kim Y.
      • Park J.
      • Sheen S.
      • Lee J.
      • Kang D.
      • Park K.
      Serum CXCR3 ligands as biomarkers for the diagnosis and treatment monitoring of tuberculosis.
      ]. Thus, these chemokines could be potentially used in several granulomatous diseases to assess prognosis and treatment efficacy.
      This study was not designed to understand the mechanisms for why these chemokines relate to different clinical endpoints. However, to explore ideas for why CXCL10 was more predictive for lung outcomes and CXCL9 was more correlated with systemic organ involvement, we compared serum protein levels to their blood mRNA transcript levels. CXCL10 protein was more strongly correlated with CXCL10 mRNA transcript level as well as with monocyte levels in the blood, while CXCL9 was not correlated with its respective mRNA transcript or monocyte levels. Prior studies have shown that both monocytes and macrophages can express CXCL9 and CXCL10 gene transcripts [
      • Proost P.
      • Vynckier A.K.
      • Mahieu F.
      • Put W.
      • Grillet B.
      • Struyf S.
      • Wuyts A.
      • Opdenakker G.
      • Van Damme J.
      Microbial Toll-like receptor ligands differentially regulate CXCL10/IP-10 expression in fibroblasts and mononuclear leukocytes in synergy with IFN-gamma and provide a mechanism for enhanced synovial chemokine levels in septic arthritis.
      ,
      • Zipfel P.F.
      • Bialonski A.
      • Skerka C.
      Induction of members of the IL-8/NAP-1 gene family in human T lymphocytes is suppressed by cyclosporin A.
      ,
      • Van Raemdonck K.
      • Van den Steen P.E.
      • Liekens S.
      • Van Damme J.
      • Struyf S.
      CXCR3 ligands in disease and therapy.
      ,
      • Farber J.M.
      A macrophage mRNA selectively induced by gamma-interferon encodes a member of the platelet factor 4 family of cytokines.
      ,
      • Loos T.
      • Dekeyzer L.
      • Struyf S.
      • Schutyser E.
      • Gijsbers K.
      • Gouwy M.
      • Fraeyman A.
      • Put W.
      • Ronsse I.
      • Grillet B.
      • Opdenakker G.
      • Van Damme J.
      • Proost P.
      TLR ligands and cytokines induce CXCR3 ligands in endothelial cells: enhanced CXCL9 in autoimmune arthritis.
      ], although human monocytes cultured ex vivo have shown greater secretion of CXCL10 protein compared to CXCL9 after stimulation with IFN-α2a [
      • Padovan E.
      • Spagnoli G.C.
      • Ferrantini M.
      • Heberer M.
      IFN-alpha2a induces IP-10/CXCL10 and MIG/CXCL9 production in monocyte-derived dendritic cells and enhances their capacity to attract and stimulate CD8+ effector T cells.
      ]. Given that our data are observational, we can only speculate on potential explanations for our findings in the context of the clinical observations we found. One possibility is that major sources of CXCL10 protein in the blood are peripheral circulating monocytes and cells in the lung, while blood CXCL9 protein levels are influenced more by activities of IFN-γ on fibroblasts, endothelial cells, and macrophages in affected tissues throughout the body. Future understanding of the cellular regulation of these chemokines in vivo will further our understanding of the roles of these proteins in the disease pathogenesis.
      Our study was limited by the lack of longitudinal data related to organ involvement and chest radiography, which prevented us from assessing these outcomes over time. The fact that we did not find an association between these chemokines and fibrosis on chest imaging is likely due to a combination of low power (only 22% of subjects had fibrosis), the lack of serial imaging to identify those who may have developed fibrosis during the follow-up period, and the lack of PET scan imaging to differentiate those subjects with fibrosis who do not have evidence of granulomatous inflammation from those with persistent inflammation. To address the dropout in our cohort, we used mixed effects modeling methodology for our longitudinal analyses, which allowed us to account for variable follow-up [
      • McLean R.A.
      • Sanders W.L.
      • Stroup W.W.
      A unified approach to mixed linear models.
      ]. Another important limitation relates to generalizability since our cohort was heterogeneous, therefore our study design did not allow us to extrapolate the prognostic value CXCL10 or CXCL9 levels at initial diagnosis and is also was not designed to address the question of whether these chemokine levels can predict the likelihood of spontaneous remission. Additionally, while we did not find any differences in CXCL9 or CXCL10 based on race, our cohort is demographically composed of greater numbers of white subjects than other racial or ethnic groups. Some of these limitations can be addressed in future studies that take advantage of existing biorepositories from other large U.S.-based cohorts such as those from the GRADS and ACCESS studies [
      • Baughman R.P.
      • Teirstein A.S.
      • Judson M.A.
      • Rossman M.D.
      • Yeager Jr., H.
      • Bresnitz E.A.
      • DePalo L.
      • Hunninghake G.
      • Iannuzzi M.C.
      • Johns C.J.
      • McLennan G.
      • Moller D.R.
      • Newman L.S.
      • Rabin D.L.
      • Rose C.
      • Rybicki B.
      • Weinberger S.E.
      • Terrin M.L.
      • Knatterud G.L.
      • Cherniak R.
      Clinical characteristics of patients in a case control study of sarcoidosis.
      ,
      • Moller D.R.
      • Koth L.L.
      • Maier L.A.
      • Morris A.
      • Drake W.
      • Rossman M.
      • Leader J.K.
      • Collman R.G.
      • Hamzeh N.
      • Sweiss N.J.
      • Zhang Y.
      • O'Neal S.
      • Senior R.M.
      • Becich M.
      • Hochheiser H.S.
      • Kaminski N.
      • Wisniewski S.R.
      • Gibson K.F.
      Rationale and design of the genomic research in alpha-1 antitrypsin deficiency and sarcoidosis (GRADS) study. Sarcoidosis protocol.
      ,
      • Judson M.A.
      • Baughman R.P.
      • Thompson B.W.
      • Teirstein A.S.
      • Terrin M.L.
      • Rossman M.D.
      • Yeager Jr., H.
      • McLennan G.
      • Bresnitz E.A.
      • DePalo L.
      • Hunninghake G.
      • Iannuzzi M.C.
      • Johns C.J.
      • Moller D.R.
      • Newman L.S.
      • Rabin D.L.
      • Rose C.
      • Rybicki B.A.
      • Weinberger S.E.
      • Knatterud G.L.
      • Cherniak R.
      Two year prognosis of sarcoidosis: the ACCESS experience. Sarcoidosis, vasculitis, and diffuse lung diseases.
      ].

      5. Conclusions and future directions

      In summary, we provide evidence showing that serum CXCL10 levels correlated with a greater number of lung function measures, pulmonary function decline, and respiratory symptoms as compared to CXCL9, which had greater correlation with systemic organ involvement. These differences may be related to each chemokine's cellular source, which is supported by our analyses using levels of mRNA transcripts and circulating immune cells. Future goals include determining how CXCL9 and CXCL10 correlate with outcomes when measured at time of diagnosis and how they change in response to treatment. With this information, we may be able to leverage biological data taken at the time of sarcoidosis diagnosis to inform patient prognosis and guide clinical decision making.

      Funding

      This work was supported by the National Institutes of Health ( R56IO87652 and T32HL007185 ).

      Ethical approval

      All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

      Informed consent

      Informed consent was obtained from all individual participants included in the study.

      Research involving animal studies

      This article does not contain any studies with animals performed by any of the authors.

      Declaration of competing interest

      All the other authors declare that they have no conflict of interest.

      Acknowledgements

      The authors thank the following individuals for their specific contributions: Michelle Nguyen, Joris Ramstein, Christine Nguyen, Sara Sun, and Zoe Lehman for assistance with sample acquisition, analysis, and management of the database; and Owen Solberg, Ph.D., for database programming. We would also like to thank all of the participants who volunteered their time for this study.

      Appendix A. Supplementary data

      The following are the Supplementary data to this article:

      References

        • Chappell A.G.
        • Cheung W.Y.
        • Hutchings H.A.
        Sarcoidosis: a long-term follow up study. Sarcoidosis, vasculitis, and diffuse lung diseases.
        official journal of WASOG. 2000; 17: 167-173
        • Iannuzzi M.C.
        • Rybicki B.A.
        • Teirstein A.S.
        Sarcoidosis.
        N. Engl. J. Med. 2007; 357: 2153-2165https://doi.org/10.1056/NEJMra071714
        • Rabin D.L.
        • Richardson M.S.
        • Stein S.R.
        • Yeager Jr., H.
        Sarcoidosis severity and socioeconomic status.
        Eur. Respir. J. 2001; 18: 499-506
        • Hunninghake G.W.
        • Costabel U.
        • Ando M.
        • Baughman R.
        • Cordier J.F.
        • du Bois R.
        • Eklund A.
        • Kitaichi M.
        • Lynch J.
        • Rizzato G.
        • Rose C.
        • Selroos O.
        • Semenzato G.
        • Sharma O.P.
        ATS/ERS/WASOG statement on sarcoidosis. American thoracic society/European respiratory society/world association of sarcoidosis and other granulomatous disorders. Sarcoidosis, vasculitis, and diffuse lung diseases.
        official journal of WASOG. 1999; 16: 149-173
        • Gerke A.K.
        Morbidity and mortality in sarcoidosis.
        Curr. Opin. Pulm. Med. 2014; 20: 472-478https://doi.org/10.1097/mcp.0000000000000080
        • Gerke A.K.
        • Judson M.A.
        • Cozier Y.C.
        • Culver D.A.
        • Koth L.L.
        Disease burden and variability in sarcoidosis.
        Annals of the American Thoracic Society. 2017; 14 (S421-s8)https://doi.org/10.1513/AnnalsATS.201707-564OT
        • Dwyer-Lindgren L.
        • Bertozzi-Villa A.
        • Stubbs R.W.
        • Morozoff C.
        • Shirude S.
        • Naghavi M.
        • Mokdad A.H.
        • Murray C.J.L.
        Trends and patterns of differences in chronic respiratory disease mortality among US counties.
        Jama. 2017; 318 (1980-2014): 1136-1149https://doi.org/10.1001/jama.2017.11747
        • Robinson B.W.
        • McLemore T.L.
        • Crystal R.G.
        Gamma interferon is spontaneously released by alveolar macrophages and lung T lymphocytes in patients with pulmonary sarcoidosis.
        J. Clin. Investig. 1985; 75: 1488-1495https://doi.org/10.1172/JCI111852
        • Prasse A.
        • Georges C.G.
        • Biller H.
        • Hamm H.
        • Matthys H.
        • Luttmann W.
        • Virchow J.C.
        Th1 cytokine pattern in sarcoidosis is expressed by bronchoalveolar CD4(+) and CD8(+) T cells.
        Clin. Exp. Immunol. 2000; 122: 241-248https://doi.org/10.1046/j.1365-2249.2000.01365.x
        • Inui N.
        • Chida K.
        • Suda T.
        • Nakamura H.
        TH1/TH2 and TC1/TC2 profiles in peripheral blood and bronchoalveolar lavage fluid cells in pulmonary sarcoidosis.
        J. Allergy Clin. Immunol. 2001; 107: 337-344https://doi.org/10.1067/mai.2001.112273
        • Mollers M.
        • Aries S.P.
        • Dromann D.
        • Mascher B.
        • Braun J.
        • Dalhoff K.
        Intracellular cytokine repertoire in different T cell subsets from patients with sarcoidosis.
        Thorax. 2001; 56: 487-493
        • Wahlstrom J.
        • Katchar K.
        • Wigzell H.
        • Olerup O.
        • Eklund A.
        • Grunewald J.
        Analysis of intracellular cytokines in CD4+ and CD8+ lung and blood T cells in sarcoidosis.
        Am. J. Respir. Crit. Care Med. 2001; 163: 115-121https://doi.org/10.1164/ajrccm.163.1.9906071
        • Kriegova E.
        • Fillerova R.
        • Tomankova T.
        • Hutyrova B.
        • Mrazek F.
        • Tichy T.
        • Kolek V.
        • du Bois R.M.
        • Petrek M.
        T-helper cell type-1 transcription factor T-bet is upregulated in pulmonary sarcoidosis.
        Eur. Respir. J. 2011; 38: 1136-1144https://doi.org/10.1183/09031936.00089910
        • Antoniou K.M.
        • Tzouvelekis A.
        • Alexandrakis M.G.
        • Sfiridaki K.
        • Tsiligianni I.
        • Rachiotis G.
        • Tzanakis N.
        • Bouros D.
        • Milic-Emili J.
        • Siafakas N.M.
        Different angiogenic activity in pulmonary sarcoidosis and idiopathic pulmonary fibrosis.
        Chest. 2006; 130: 982-988https://doi.org/10.1378/chest.130.4.982
        • Sugiyama K.
        • Mukae H.
        • Ishii H.
        • Kakugawa T.
        • Ishimoto H.
        • Nakayama S.
        • Shirai R.
        • Fujii T.
        • Mizuta Y.
        • Kohno S.
        Elevated levels of interferon gamma-inducible protein-10 and epithelial neutrophil-activating peptide-78 in patients with pulmonary sarcoidosis.
        Respirology. 2006; 11: 708-714https://doi.org/10.1111/j.1440-1843.2006.00933.x
        • Nishioka Y.
        • Manabe K.
        • Kishi J.
        • Wang W.
        • Inayama M.
        • Azuma M.
        • Sone S.
        CXCL9 and 11 in patients with pulmonary sarcoidosis: a role of alveolar macrophages.
        Clin. Exp. Immunol. 2007; 149: 317-326https://doi.org/10.1111/j.1365-2249.2007.03423.x
        • Nureki S.
        • Miyazaki E.
        • Ando M.
        • Ueno T.
        • Fukami T.
        • Kumamoto T.
        • Sugisaki K.
        • Tsuda T.
        Circulating levels of both Th1 and Th2 chemokines are elevated in patients with sarcoidosis.
        Respir. Med. 2008; 102: 239-247https://doi.org/10.1016/j.rmed.2007.09.006
        • Nagata K.
        • Maruyama K.
        • Uno K.
        • Shinomiya K.
        • Yoneda K.
        • Hamuro J.
        • Sugita S.
        • Yoshimura T.
        • Sonoda K.H.
        • Mochizuki M.
        • Kinoshita S.
        Simultaneous analysis of multiple cytokines in the vitreous of patients with sarcoid uveitis.
        Investig. Ophthalmol. Vis. Sci. 2012; 53: 3827-3833https://doi.org/10.1167/iovs.11-9244
        • Takeuchi M.
        • Oh I.K.
        • Suzuki J.
        • Hattori T.
        • Takeuchi A.
        • Okunuki Y.
        • Usui Y.
        • Usui M.
        Elevated serum levels of CXCL9/monokine induced by interferon-gamma and CXCL10/interferon-gamma-inducible protein-10 in ocular sarcoidosis.
        Investig. Ophthalmol. Vis. Sci. 2006; 47: 1063-1068https://doi.org/10.1167/iovs.05-0966
        • Koth L.L.
        • Solberg O.D.
        • Peng J.C.
        • Bhakta N.R.
        • Nguyen C.P.
        • Woodruff P.G.
        Sarcoidosis blood transcriptome reflects lung inflammation and overlaps with tuberculosis.
        Am. J. Respir. Crit. Care Med. 2011; 184: 1153-1163https://doi.org/10.1164/rccm.201106-1143OC
        • Su R.
        • Nguyen M.L.
        • Agarwal M.R.
        • Kirby C.
        • Nguyen C.P.
        • Ramstein J.
        • Darnell E.P.
        • Gomez A.D.
        • Ho M.
        • Woodruff P.G.
        • Koth L.L.
        Interferon-inducible chemokines reflect severity and progression in sarcoidosis.
        Respir. Res. 2013; 14: 121https://doi.org/10.1186/1465-9921-14-121
        • Su R.
        • Li M.M.
        • Bhakta N.R.
        • Solberg O.D.
        • Darnell E.P.
        • Ramstein J.
        • Garudadri S.
        • Ho M.
        • Woodruff P.G.
        • Koth L.L.
        Longitudinal analysis of sarcoidosis blood transcriptomic signatures and disease outcomes.
        Eur. Respir. J. 2014; 44: 985-993https://doi.org/10.1183/09031936.00039714
        • Arger N.K.
        • Ho M.
        • Woodruff P.G.
        • Koth L.L.
        Serum CXCL11 correlates with pulmonary outcomes and disease burden in sarcoidosis.
        Respir. Med. 2019; 152: 89-96https://doi.org/10.1016/j.rmed.2019.04.005
        • Groom J.R.
        • Luster A.D.
        CXCR3 ligands: redundant, collaborative and antagonistic functions.
        Immunol. Cell Biol. 2011; 89: 207-215https://doi.org/10.1038/icb.2010.158
        • Grimm M.C.
        • Doe W.F.
        Chemokines in inflammatory bowel disease mucosa: expression of RANTES, macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, and gamma-interferon-inducible protein-10 by macrophages, lymphocytes, endothelial cells, and granulomas.
        Inflamm. Bowel Dis. 1996; 2: 88-96
        • Kishi J.
        • Nishioka Y.
        • Kuwahara T.
        • Kakiuchi S.
        • Azuma M.
        • Aono Y.
        • Makino H.
        • Kinoshita K.
        • Kishi M.
        • Batmunkh R.
        • Uehara H.
        • Izumi K.
        • Sone S.
        Blockade of Th1 chemokine receptors ameliorates pulmonary granulomatosis in mice.
        Eur. Respir. J. 2011; 38: 415-424https://doi.org/10.1183/09031936.00070610
        • Aranday-Cortes E.
        • Bull N.C.
        • Villarreal-Ramos B.
        • Gough J.
        • Hicks D.
        • Ortiz-Pelaez A.
        • Vordermeier H.M.
        • Salguero F.J.
        Upregulation of IL-17A, CXCL9 and CXCL10 in early-stage granulomas induced by Mycobacterium bovis in cattle.
        Transboundary and emerging diseases. 2013; 60: 525-537https://doi.org/10.1111/j.1865-1682.2012.01370.x
        • Torraca V.
        • Cui C.
        • Boland R.
        • Bebelman J.P.
        • van der Sar A.M.
        • Smit M.J.
        • Siderius M.
        • Spaink H.P.
        • Meijer A.H.
        The CXCR3-CXCL11 signaling axis mediates macrophage recruitment and dissemination of mycobacterial infection.
        Disease models & mechanisms. 2015; 8: 253-269https://doi.org/10.1242/dmm.017756
        • Agostini C.
        • Cassatella M.
        • Zambello R.
        • Trentin L.
        • Gasperini S.
        • Perin A.
        • Piazza F.
        • Siviero M.
        • Facco M.
        • Dziejman M.
        • Chilosi M.
        • Qin S.
        • Luster A.D.
        • Semenzato G.
        Involvement of the IP-10 chemokine in sarcoid granulomatous reactions.
        J. Immunol. 1998; 161: 6413-6420
        • Aksoy M.O.
        • Yang Y.
        • Ji R.
        • Reddy P.J.
        • Shahabuddin S.
        • Litvin J.
        • Rogers T.J.
        • Kelsen S.G.
        CXCR3 surface expression in human airway epithelial cells: cell cycle dependence and effect on cell proliferation.
        Am. J. Physiol. Lung Cell Mol. Physiol. 2006; 290: L909-L918https://doi.org/10.1152/ajplung.00430.2005
        • Strieter R.M.
        • Burdick M.D.
        • Gomperts B.N.
        • Belperio J.A.
        • Keane M.P.
        CXC chemokines in angiogenesis.
        Cytokine Growth Factor Rev. 2005; 16: 593-609https://doi.org/10.1016/j.cytogfr.2005.04.007
        • Liu L.
        • Callahan M.K.
        • Huang D.
        • Ransohoff R.M.
        Chemokine receptor CXCR3: an unexpected enigma.
        Curr. Top. Dev. Biol. 2005; 68: 149-181https://doi.org/10.1016/s0070-2153(05)68006-4
        • Hancock W.W.
        • Gao W.
        • Csizmadia V.
        • Faia K.L.
        • Shemmeri N.
        • Luster A.D.
        Donor-derived IP-10 initiates development of acute allograft rejection.
        J. Exp. Med. 2001; 193: 975-980https://doi.org/10.1084/jem.193.8.975
        • Burns J.M.
        • Summers B.C.
        • Wang Y.
        • Melikian A.
        • Berahovich R.
        • Miao Z.
        • Penfold M.E.
        • Sunshine M.J.
        • Littman D.R.
        • Kuo C.J.
        • Wei K.
        • McMaster B.E.
        • Wright K.
        • Howard M.C.
        • Schall T.J.
        A novel chemokine receptor for SDF-1 and I-TAC involved in cell survival, cell adhesion, and tumor development.
        J. Exp. Med. 2006; 203: 2201-2213https://doi.org/10.1084/jem.20052144
        • Zohar Y.
        • Wildbaum G.
        • Novak R.
        • Salzman A.L.
        • Thelen M.
        • Alon R.
        • Barsheshet Y.
        • Karp C.L.
        • Karin N.
        CXCL11-dependent induction of FOXP3-negative regulatory T cells suppresses autoimmune encephalomyelitis.
        J. Clin. Investig. 2014; 124: 2009-2022https://doi.org/10.1172/JCI71951
        • Colvin R.A.
        • Campanella G.S.
        • Sun J.
        • Luster A.D.
        Intracellular domains of CXCR3 that mediate CXCL9, CXCL10, and CXCL11 function.
        J. Biol. Chem. 2004; 279: 30219-30227https://doi.org/10.1074/jbc.M403595200
        • Cole K.E.
        • Strick C.A.
        • Paradis T.J.
        • Ogborne K.T.
        • Loetscher M.
        • Gladue R.P.
        • Lin W.
        • Boyd J.G.
        • Moser B.
        • Wood D.E.
        • Sahagan B.G.
        • Neote K.
        Interferon-inducible T cell alpha chemoattractant (I-TAC): a novel non-ELR CXC chemokine with potent activity on activated T cells through selective high affinity binding to CXCR3.
        J. Exp. Med. 1998; 187: 2009-2021https://doi.org/10.1084/jem.187.12.2009
        • Xanthou G.
        • Duchesnes C.E.
        • Williams T.J.
        • Pease J.E.
        CCR3 functional responses are regulated by both CXCR3 and its ligands CXCL9, CXCL10 and CXCL11.
        Eur. J. Immunol. 2003; 33: 2241-2250https://doi.org/10.1002/eji.200323787
        • Wang Q.
        • Nagarkar D.R.
        • Bowman E.R.
        • Schneider D.
        • Gosangi B.
        • Lei J.
        • Zhao Y.
        • McHenry C.L.
        • Burgens R.V.
        • Miller D.J.
        • Sajjan U.
        • Hershenson M.B.
        Role of double-stranded RNA pattern recognition receptors in rhinovirus-induced airway epithelial cell responses.
        J. Immunol. 2009; 183: 6989-6997https://doi.org/10.4049/jimmunol.0901386
        • Amichay D.
        • Gazzinelli R.T.
        • Karupiah G.
        • Moench T.R.
        • Sher A.
        • Farber J.M.
        Genes for chemokines MuMig and Crg-2 are induced in protozoan and viral infections in response to IFN-gamma with patterns of tissue expression that suggest nonredundant roles in vivo.
        J. Immunol. 1996; 157: 4511-4520
        • Nakanishi Y.
        • Lu B.
        • Gerard C.
        • Iwasaki A.
        CD8(+) T lymphocyte mobilization to virus-infected tissue requires CD4(+) T-cell help.
        Nature. 2009; 462: 510-513https://doi.org/10.1038/nature08511
        • Medoff B.D.
        • Wain J.C.
        • Seung E.
        • Jackobek R.
        • Means T.K.
        • Ginns L.C.
        • Farber J.M.
        • Luster A.D.
        CXCR3 and its ligands in a murine model of obliterative bronchiolitis: regulation and function.
        J. Immunol. 2006; 176: 7087-7095https://doi.org/10.4049/jimmunol.176.11.7087
        • Ohmori Y.
        • Wyner L.
        • Narumi S.
        • Armstrong D.
        • Stoler M.
        • Hamilton T.A.
        Tumor necrosis factor-alpha induces cell type and tissue-specific expression of chemoattractant cytokines in vivo.
        Am. J. Pathol. 1993; 142: 861-870
        • Ciesielski C.J.
        • Andreakos E.
        • Foxwell B.M.
        • Feldmann M.
        TNFalpha-induced macrophage chemokine secretion is more dependent on NF-kappaB expression than lipopolysaccharides-induced macrophage chemokine secretion.
        Eur. J. Immunol. 2002; 32: 2037-2045https://doi.org/10.1002/1521-4141(200207)32:7<2037::aid-immu2037>3.0.co;2-i
        • Luster A.D.
        • Unkeless J.C.
        • Ravetch J.V.
        Gamma-interferon transcriptionally regulates an early-response gene containing homology to platelet proteins.
        Nature. 1985; 315: 672-676https://doi.org/10.1038/315672a0
        • Proost P.
        • Verpoest S.
        • Van de Borne K.
        • Schutyser E.
        • Struyf S.
        • Put W.
        • Ronsse I.
        • Grillet B.
        • Opdenakker G.
        • Van Damme J.
        Synergistic induction of CXCL9 and CXCL11 by Toll-like receptor ligands and interferon-gamma in fibroblasts correlates with elevated levels of CXCR3 ligands in septic arthritis synovial fluids.
        J. Leukoc. Biol. 2004; 75: 777-784https://doi.org/10.1189/jlb.1003524
        • Proost P.
        • Vynckier A.K.
        • Mahieu F.
        • Put W.
        • Grillet B.
        • Struyf S.
        • Wuyts A.
        • Opdenakker G.
        • Van Damme J.
        Microbial Toll-like receptor ligands differentially regulate CXCL10/IP-10 expression in fibroblasts and mononuclear leukocytes in synergy with IFN-gamma and provide a mechanism for enhanced synovial chemokine levels in septic arthritis.
        Eur. J. Immunol. 2003; 33: 3146-3153https://doi.org/10.1002/eji.200324136
        • Benn B.S.
        • Lehman Z.
        • Kidd S.A.
        • Ho M.
        • Sun S.
        • Ramstein J.
        • Arger N.K.
        • Nguyen C.P.
        • Su R.
        • Gomez A.
        • Gelfand J.M.
        • Koth L.L.
        Clinical and biological insights from the university of California san Francisco prospective and longitudinal cohort.
        Lung. 2017; https://doi.org/10.1007/s00408-017-0037-y
        • Eakin E.G.
        • Resnikoff P.M.
        • Prewitt L.M.
        • Ries A.L.
        • Kaplan R.M.
        Validation of a new dyspnea measure: the UCSD shortness of breath questionnaire.
        Chest. 1998; 113 (University of California, San Diego): 619-624
        • Swigris J.J.
        • Yorke J.
        • Sprunger D.B.
        • Swearingen C.
        • Pincus T.
        • du Bois R.M.
        • Brown K.K.
        • Fischer A.
        Assessing dyspnea and its impact on patients with connective tissue disease-related interstitial lung disease.
        Respir. Med. 2010; 104: 1350-1355https://doi.org/10.1016/j.rmed.2010.03.027
        • Swigris J.J.
        • Han M.
        • Vij R.
        • Noth I.
        • Eisenstein E.L.
        • Anstrom K.J.
        • Brown K.K.
        • Fairclough D.
        The UCSD shortness of breath questionnaire has longitudinal construct validity in idiopathic pulmonary fibrosis.
        Respir. Med. 2012; 106: 1447-1455https://doi.org/10.1016/j.rmed.2012.06.018
        • Vittinghoff E.
        • Glidden D.V.
        • Shiboski S.C.
        • McCulloch C.E.
        Statistics for Biology and Health.
        2012: 7-26
        • McLean R.A.
        • Sanders W.L.
        • Stroup W.W.
        A unified approach to mixed linear models.
        Am. Stat. 1991; 45: 54-64https://doi.org/10.1080/00031305.1991.10475767
        • Keir G.
        • Wells A.U.
        Assessing pulmonary disease and response to therapy: which test?.
        Semin. Respir. Crit. Care Med. 2010; 31: 409-418https://doi.org/10.1055/s-0030-1262209
        • Baughman R.P.
        • Teirstein A.S.
        • Judson M.A.
        • Rossman M.D.
        • Yeager Jr., H.
        • Bresnitz E.A.
        • DePalo L.
        • Hunninghake G.
        • Iannuzzi M.C.
        • Johns C.J.
        • McLennan G.
        • Moller D.R.
        • Newman L.S.
        • Rabin D.L.
        • Rose C.
        • Rybicki B.
        • Weinberger S.E.
        • Terrin M.L.
        • Knatterud G.L.
        • Cherniak R.
        Clinical characteristics of patients in a case control study of sarcoidosis.
        Am. J. Respir. Crit. Care Med. 2001; 164: 1885-1889https://doi.org/10.1164/ajrccm.164.10.2104046
        • Schoenfeld D.
        Partial residuals for the proportional hazards regression model.
        Biometrika. 1982; 69: 239-241https://doi.org/10.2307/2335876
        • Snijders T.A.B.
        • Bosker R.J.
        Modeled variance in two-level models.
        Sociol. Methods Res. 1994; 22: 342-363https://doi.org/10.1177/0049124194022003004
        • Luster A.D.
        • Ravetch J.V.
        Biochemical characterization of a gamma interferon-inducible cytokine (IP-10).
        J. Exp. Med. 1987; 166: 1084-1097
        • Zipfel P.F.
        • Bialonski A.
        • Skerka C.
        Induction of members of the IL-8/NAP-1 gene family in human T lymphocytes is suppressed by cyclosporin A.
        Biochem. Biophys. Res. Commun. 1991; 181: 179-183
        • Padovan E.
        • Spagnoli G.C.
        • Ferrantini M.
        • Heberer M.
        IFN-alpha2a induces IP-10/CXCL10 and MIG/CXCL9 production in monocyte-derived dendritic cells and enhances their capacity to attract and stimulate CD8+ effector T cells.
        J. Leukoc. Biol. 2002; 71: 669-676
        • Prior C.
        • Haslam P.L.
        Increased levels of serum interferon-gamma in pulmonary sarcoidosis and relationship with response to corticosteroid therapy.
        Am. Rev. Respir. Dis. 1991; 143: 53-60https://doi.org/10.1164/ajrccm/143.1.53
        • Miotto D.
        • Christodoulopoulos P.
        • Olivenstein R.
        • Taha R.
        • Cameron L.
        • Tsicopoulos A.
        • Tonnel A.B.
        • Fahy O.
        • Lafitte J.J.
        • Luster A.D.
        • Wallaert B.
        • Mapp C.E.
        • Hamid Q.
        Expression of IFN-gamma-inducible protein; monocyte chemotactic proteins 1, 3, and 4; and eotaxin in TH1- and TH2-mediated lung diseases.
        J. Allergy Clin. Immunol. 2001; 107 (S0091-6749(01)23352-1 [pii]): 664-670
        • Cui A.
        • Anhenn O.
        • Theegarten D.
        • Ohshimo S.
        • Bonella F.
        • Sixt S.U.
        • Peters J.
        • Sarria R.
        • Guzman J.
        • Costabel U.
        Angiogenic and angiostatic chemokines in idiopathic pulmonary fibrosis and granulomatous lung disease.
        Respiration; international review of thoracic diseases. 2010; 80: 372-378https://doi.org/10.1159/000245332
        • Arakelyan A.
        • Kriegova E.
        • Kubistova Z.
        • Mrazek F.
        • Kverka M.
        • du Bois R.M.
        • Kolek V.
        • Petrek M.
        Protein levels of CC chemokine ligand (CCL)15, CCL16 and macrophage stimulating protein in patients with sarcoidosis.
        Clin. Exp. Immunol. 2009; 155: 457-465https://doi.org/10.1111/j.1365-2249.2008.03832.x
        • Piotrowski W.J.
        • Mlynarski W.
        • Fendler W.
        • Wyka K.
        • Marczak J.
        • Gorski P.
        • Antczak A.
        Chemokine receptor CXCR3 ligands in bronchoalveolar lavage fluid: associations with radiological pattern, clinical course, and prognosis in sarcoidosis.
        Polskie Arch. Med. Wewn. 2014; 124: 395-402
        • Li H.
        • Zhao X.
        • Wang J.
        • Zong M.
        • Yang H.
        Bioinformatics analysis of gene expression profile data to screen key genes involved in pulmonary sarcoidosis.
        Gene. 2017; 596: 98-104https://doi.org/10.1016/j.gene.2016.09.037
        • Pignatti P.
        • Brunetti G.
        • Moretto D.
        • Yacoub M.R.
        • Fiori M.
        • Balbi B.
        • Balestrino A.
        • Cervio G.
        • Nava S.
        • Moscato G.
        Role of the chemokine receptors CXCR3 and CCR4 in human pulmonary fibrosis.
        Am. J. Respir. Crit. Care Med. 2006; 173: 310-317https://doi.org/10.1164/rccm.200502-244OC
        • Busuttil A.
        • Weigt S.S.
        • Keane M.P.
        • Xue Y.Y.
        • Palchevskiy V.
        • Burdick M.D.
        • Huang C.
        • Zisman D.A.
        • Fishbein M.
        • Lynch 3rd, J.P.
        • Strieter R.M.
        • Elashoff R.M.
        • Belperio J.A.
        CXCR3 ligands are augmented during the pathogenesis of pulmonary sarcoidosis.
        Eur. Respir. J. 2009; 34: 676-686https://doi.org/10.1183/09031936.00157508
        • Schnerch J.
        • Prasse A.
        • Vlachakis D.
        • Schuchardt K.L.
        • Pechkovsky D.V.
        • Goldmann T.
        • Gaede K.I.
        • Muller-Quernheim J.
        • Zissel G.
        Functional toll-like receptor 9 expression and CXCR3 ligand release in pulmonary sarcoidosis.
        Am. J. Respir. Cell Mol. Biol. 2016; 55: 749-757https://doi.org/10.1165/rcmb.2015-0278OC
        • Kim S.
        • Lee H.
        • Kim H.
        • Kim Y.
        • Cho J.E.
        • Jin H.
        • Kim D.Y.
        • Ha S.J.
        • Kang Y.A.
        • Cho S.N.
        • Lee H.
        Diagnostic performance of a cytokine and IFN-gamma-induced chemokine mRNA assay after Mycobacterium tuberculosis-specific antigen stimulation in whole blood from infected individuals.
        J. Mol. Diagn. : J. Mod. Dyn. 2015; 17: 90-99https://doi.org/10.1016/j.jmoldx.2014.08.005
        • Lee K.
        • Chung W.
        • Jung Y.
        • Kim Y.
        • Park J.
        • Sheen S.
        • Park K.
        CXCR3 ligands as clinical markers for pulmonary tuberculosis.
        Int. J. Tuberc. Lung Dis. : the official journal of the International Union against Tuberculosis and Lung Disease. 2015; 19: 191-199https://doi.org/10.5588/ijtld.14.0525
        • Chung W.
        • Lee K.
        • Jung Y.
        • Kim Y.
        • Park J.
        • Sheen S.
        • Lee J.
        • Kang D.
        • Park K.
        Serum CXCR3 ligands as biomarkers for the diagnosis and treatment monitoring of tuberculosis.
        Int. J. Tuberc. Lung Dis. : the official journal of the International Union against Tuberculosis and Lung Disease. 2015; 19: 1476-1484https://doi.org/10.5588/ijtld.15.0325
        • Van Raemdonck K.
        • Van den Steen P.E.
        • Liekens S.
        • Van Damme J.
        • Struyf S.
        CXCR3 ligands in disease and therapy.
        Cytokine Growth Factor Rev. 2015; 26: 311-327https://doi.org/10.1016/j.cytogfr.2014.11.009
        • Farber J.M.
        A macrophage mRNA selectively induced by gamma-interferon encodes a member of the platelet factor 4 family of cytokines.
        in: Proceedings of the National Academy of Sciences of the United States of America. vol. 87. 1990: 5238-5242https://doi.org/10.1073/pnas.87.14.5238 (14)
        • Loos T.
        • Dekeyzer L.
        • Struyf S.
        • Schutyser E.
        • Gijsbers K.
        • Gouwy M.
        • Fraeyman A.
        • Put W.
        • Ronsse I.
        • Grillet B.
        • Opdenakker G.
        • Van Damme J.
        • Proost P.
        TLR ligands and cytokines induce CXCR3 ligands in endothelial cells: enhanced CXCL9 in autoimmune arthritis.
        Laboratory investigation; a journal of technical methods and pathology. 2006; 86: 902-916https://doi.org/10.1038/labinvest.3700453
        • Moller D.R.
        • Koth L.L.
        • Maier L.A.
        • Morris A.
        • Drake W.
        • Rossman M.
        • Leader J.K.
        • Collman R.G.
        • Hamzeh N.
        • Sweiss N.J.
        • Zhang Y.
        • O'Neal S.
        • Senior R.M.
        • Becich M.
        • Hochheiser H.S.
        • Kaminski N.
        • Wisniewski S.R.
        • Gibson K.F.
        Rationale and design of the genomic research in alpha-1 antitrypsin deficiency and sarcoidosis (GRADS) study. Sarcoidosis protocol.
        Annals of the American Thoracic Society. 2015; 12: 1561-1571https://doi.org/10.1513/AnnalsATS.201503-172OT
        • Judson M.A.
        • Baughman R.P.
        • Thompson B.W.
        • Teirstein A.S.
        • Terrin M.L.
        • Rossman M.D.
        • Yeager Jr., H.
        • McLennan G.
        • Bresnitz E.A.
        • DePalo L.
        • Hunninghake G.
        • Iannuzzi M.C.
        • Johns C.J.
        • Moller D.R.
        • Newman L.S.
        • Rabin D.L.
        • Rose C.
        • Rybicki B.A.
        • Weinberger S.E.
        • Knatterud G.L.
        • Cherniak R.
        Two year prognosis of sarcoidosis: the ACCESS experience. Sarcoidosis, vasculitis, and diffuse lung diseases.
        official journal of WASOG. 2003; 20: 204-211