Scand J Rheumatol 2014;43:403–408

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Decreased tumour necrosis factor-α production by monocytes of granulomatosis with polyangiitis JK Park, EB Lee, YW Song

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Division of Rheumatology, Department of Medicine, Seoul National University Hospital, Seoul, Korea

Objectives: We hypothesized that monocytes in patients with granulomatosis with polyangiitis (GPA) are polarized towards alternative activation with decreased tumour necrosis factor (TNF)-α production and that tissue-infiltrating monocytes/macrophages in granulomatous GPA lesions express CD163, a marker of alternative macrophage activation. Method: CD16þ monocytes in peripheral blood mononuclear cells (PBMCs) were quantified by flow cytometry. Monocytes were stimulated with increasing concentrations of lipopolysaccharide (LPS), and TNF-α production was measured at 4 and 24 h. CD163 expression in lung biopsies of patients with GPA was detected by immunohistochemistry. Results: Circulating CD16þ monocytes were more frequent in GPA patients compared to controls (4.7  2.8% vs. 1.9  1.2%, p < 0.001). Upon activation with LPS, TNF-α production did not differ between CD16þ and CD16– monocytes. Stimulated monocytes from GPA patients produced significantly less TNF-α compared with monocytes from healthy controls (2903  1320 pg/mL vs. 8335  4569 pg/mL, p < 0.001). Macrophages expressing CD163 were enriched in granulomatous lung lesions of GPA patients. Conclusions: Decreased TNF-α production by circulating monocytes and CD163 overexpression by tissue monocytes/ macrophages in granulomatous pulmonary lesions may suggest that monocytes/macrophages are alternatively activated in GPA.

Granulomatosis with polyangiitis (GPA), formerly known as Wegener’s granulomatosis, is characterized by necrotizing vasculitis and granulomatous inflammation (1). The granulomatous inflammation in GPA contains cells that differentiate from the monocyte lineage, that is macrophages, dendritic cells, epitheloid cells, and multinucleated giant cells (MNGs) (2). We showed previously that monocytes within the peripheral blood mononuclear cells (PBMCs) in patients with GPA had a higher propensity to form MNGs compared to healthy controls and that this propensity was associated with the extent of systemic inflammation (3). Of note, monocytes lose the propensity to form MNGs when activated with inflammatory stimuli such as toll-like receptor 2 and 4 agonists (4). Thus, monocytes in GPA might already be polarized towards alternative activation with a higher propensity for granulomatous inflammation at the expense of the proinflammatory response. Activated monocytes are a major source of tumour necrosis factor (TNF)-α, which promotes inflammation and aids in granuloma formation (5). Specifically,

Jin Kyun Park, Division of Rheumatology, Department of Internal Medicine, Seoul National University Hospital, 28 Yongon-dong, Chongno-gu, Seoul 110-744, Korea. E-mail: [email protected]

circulating proinflammatory monocytes, which are phenotypically defined by the surface expression of CD16, produce large amounts of TNF-α and interleukin (IL)-1 (6). These cells are in fact accumulated in both infectious and systemic autoimmune diseases including sepsis and rheumatoid arthritis (RA) (7). Accordingly, treatment directed against TNF-α has dramatically improved the prognosis of patients with RA and psoriatic arthritis (8, 9). However, the lack of efficacy of anti-TNF-α therapy for the maintenance of remission in GPA suggests that TNF-α signalling may play a less important role in this disease and that monocytes in GPA may contribute to disease progression by other mechanisms such as granulomatous inflammation (10). In GPA patients, the upper respiratory tract is heavily colonized with bacteria despite extensive local inflammation. This may indicate impaired local immunity in GPA (11, 12). Similarly, tissue-infiltrating monocytes in chronic sinusitis demonstrate an impaired immune response and express CD163 (13). Expression of CD163, a scavenger molecule, defines a subset of monocytes/macrophages that are alternatively activated and orchestrate the resolution of inflammation and wound repair (14, 15). Accordingly, we hypothesized that monocytes in GPA are alternatively activated with decreased TNF-α production and that alternatively activated CD163þ monocytes/ macrophages are abundant in granulomatous lesions of GPA patients.

Accepted 11 February 2014 © 2014 Informa Healthcare on license from Scandinavian Rheumatology Research Foundation DOI: 10.3109/03009742.2014.894568

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Method Study population GPA patients receiving clinical care at our medical centre and healthy control subjects were enrolled in the study (Supplementary Table S1). Informed consent was obtained under the auspices of an Institutional Review Board (IRB)-approved research protocol. Paraffinembedded lung tissues were obtained from the pathology archive at our institution.

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Cell isolation and activation

JK Park et al

Carpinteria, CA, USA) and CD163 (MAB1652, Abnova, Walnut, CA, USA). The staining was visualized with 3,30 -diaminobenzidine (DAB; DAKO). Nuclei were counterstained with Mayer’s haematoxylin. Monocyte culture in MNG-promoting microenvironments Isolated monocytes were cultured in OPTI-MEM® I (Gibco/Invitrogen, Grand Island, NY, USA), supplemented with 10% heat-inactivated FBS, 1% penicillin, and 1% streptomycin in the presence 100 ng/mL RANK-L (R&D Systems, Minneapolis, MN, USA) and 25 ng/mL macrophage colony-stimulating factor (M-CSF; R&D Systems) in 5% carbon dioxide at 37˚C. On days 3, 6, and 9, cells were fixed with 100% methanol and were stained with antibodies directed against CD68 and CD163. Secondary goat anti-mouse antibody conjugated with Alexa 488 and goat anti-rabbit antibody conjugated with Alexa 599 were applied for 1 h.

PBMCs were isolated from heparinized peripheral venous blood by density gradient centrifugation using Ficoll-Paque (GE Healthcare, NJ, USA). Monocytes were isolated using CD14 Microbeads according to the manufacturer’s instructions (MACS, Miltenyi Biotec, Auburn, CA, USA). PBMCs or CD14þ cells (1  106 cells per well) were activated in RPMI, supplemented with 10% heat-inactivated foetal bovine serum (FBS), 1% penicillin, and 1% streptomycin, in the presence of 0, 10, or 100 ng/mL lipopolysaccharide (LPS; InvivoGen, catalogue code tlrl-eblps, San Diego, CA, USA). Cells were incubated in 5% carbon dioxide at 37˚C for 4 or 24 h. Supernatants were collected for enzyme-linked immunosorbent assay (ELISA).

Student t-tests and Mann Whitney tests were used for continuous variables. Correlations between data were performed using Spearman correlation. p-values < 0.05 were considered significant. Statistical analyses were computed in GraphPad Prism (La Jolla, CA, USA).

Flow cytometry analysis

Results

Activated PBMCs were incubated with anti-human CD14 PerCP (BD Biosciences, San Jose, CA, USA) and antihuman CD3 antibodies (BD Biosciences) for 30 min at 4˚C. Intracellular staining of TNF-α was performed using anti-human TNF-α antibodies (BD Biosciences) according to the manufacturer’s instruction (GolgiStop, Pharmigen, San Diego, CA, USA). Cells were analysed by fluorescence-activated cell sorting on a FACSAria III (BD Biosciences). Flow data were analysed by FlowJo software version 8.8 (Treestar, Ashland, OR, USA).

CD16þ monocytes are more frequent in GPA

TNF-α ELISA TNF-α levels were measured using a commercially available ELISA kit (R&D Systems). The optical density was determined using a microplate reader (Biotrack, Pharmacia) set at 450 nm. TNF-α concentrations were extrapolated from a standard curve and expressed in pg/ mL. All samples were assayed in duplicate.

Immunohistochemistry Formalin-fixed and paraffin-embedded tissues were de-paraffinized with xylenes and rehydrated. After antigen retrieval and blocking, tissues were incubated with antibodies directed against CD68 (clone PG-MI, DAKO,

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Statistical analysis

The percentage of CD16þ monocytes in PBMCs of patients with GPA (n ¼ 22) and healthy subjects (n ¼ 14) was determined. PBMCs in GPA showed a significantly higher percentage of CD16þ monocytes than healthy controls (4.7  2.8% vs. 1.9  1.2%, p < 0.001) (Figure 1). There was no association between CD16þ monocyte levels and GPA disease activity (Spearman r ¼ –0.105, p ¼ 0.65) or disease duration (Spearman r ¼ 0.288, p ¼ 0.21). Of note, patients in our cohort had a relatively low disease activity, with a Birmingham Vasculitis Activity Score (BVAS) ranging from 0 to 5 (Supplementary Table S1).

Monocytes in GPA produce less TNF-α As M-CSF, a cytokine crucial for the generation of alternatively activated macrophages with anti-inflammatory properties (16, 17), is known to induce the expression of CD16 on monocytes, we examined whether CD16þ monocytes are more proinflammatory in GPA. Upon activation with LPS, the percentage of TNF-α-producing cells did not differ significantly in the CD16– and CD16þ monocyte fractions of both GPA patients and controls (in controls: 52.2  1.42% vs. 59.0  3.9%, p ¼ 0.06; in GPA: 44.1  16.3% vs. 52.4  10.5%, p ¼ 0.20, Figure 2A).

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Figure 1. Enrichment of CD16þ monocytes independent of disease activity in GPA. The frequency of CD16þ CD14 monocytes in PBMCs was determined from 22 GPA patients and 14 healthy controls. (A) Expression of CD16þ monocytes was significantly increased in GPA compared to healthy controls (4.8  2.8 vs. 1.0  1.2, p < 0.001). Their presence did not correlate with (B) disease activity, expressed as the Birmingham Vasculitis Activity Score (BVAS), or (C) disease duration.

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Figure 2. GPA monocytes produce a decreased amount of TNF-α upon activation. PBMCs from healthy controls (n ¼ 3) and GPA patients (n ¼ 3) were stimulated with LPS for 4 h. (A) Both CD16þ and CD16– CD14þ monocytes from healthy controls and GPA patients produced TNF-α (gated on CD14þ cells). (B) After stimulation with 10 or 100 ng/mL LPS, CD14þ monocytes from GPA produced significantly less TNF-α after 4 h. (C) Isolated CD14þ monocytes from GPA (n ¼ 3) produced and secreted less TNF-α after activation with LPS for 24 h compared to controls (n ¼ 3).

However, monocytes from patients with GPA (n ¼ 3) produced significantly less TNF-α after 4 h of stimulation with LPS than healthy controls (n ¼ 3) (for 10 ng/mL LPS: 7254  1004 MFI vs. 4015  2322 MFI, p ¼ 0.08;

for 100 ng/mL LPS: 9562  1219 MFI vs. 4855  2322 MFI, p ¼ 0.04) (Figure 2B). To quantify levels of secreted TNF-α, 2  105 isolated monocytes from healthy controls (n ¼ 3) and GPA patients (n ¼ 3) were stimulated with

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Figure 3. CD163þ monocytes/macrophages are abundant in GPA lung lesions. (A) In healthy lung tissue, alveolar macrophages expressed little to no CD163. By contrast, the monocytes/macrophages (B) in alveolar spaces and (C) in granulomatous inflammation surrounding multinucleated giant cells (MNGs) strongly expressed CD163 on the cell surface in GPA. (D) In the centre of the granulomatous inflammation only a few cells expressed CD163 and MNGs were only weakly positive for CD163. Isolated monocytes were cultured in the presence of 25 ng/ mL M-CSF and 100 ng/mL RANKL for 9 days. On day 3 (D3), CD163 (red) was expressed on the cell membrane whereas CD68 (green) showed a cytoplasmic expression pattern (E, left). On day 6 (D6), CD163 expression was weaker and small MNGs lost CD163 expression (E, centre). On day 9 (D9), only a few cells retained CD163 expression and large MNGs lacked CD163 expression (E, right). Original magnification 40. The star (*) indicates an MNG.

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LPS for 24 h. Monocytes from healthy controls produced TNF-α in a dose-dependent manner whereas the secretion of TNF-α in GPA plateaued with 10 ng/mL LPS. On stimulation with 100 ng/mL LPS, monocytes of healthy controls produced significantly more TNF-α compared to GPA patients (8335  4569 pg/mL vs. 2903  1320 pg/mL, p < 0.01) (Figure 2C).

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vicinity of the granuloma, CD163 expression was lower in macrophages and was absent in MNGs (Figure 3D). Based on these findings, we examined the pattern of CD163 expression in monocytes/macrophages during differentiation into MNGs. As monocytes differentiated into macrophages and MNGs, CD163 expression decreased, whereas CD68 expression was maintained (Figure 3E).

CD163þ cells are abundant in GPA lung granuloma We showed previously that the transcriptome of active GPA patients differs from healthy controls (18). Of note, CD163 was one of the most up-regulated transcripts in active GPA patients (unpublished data). To investigate if the inflammatory lesions in GPA reflect these peripheral transcriptional changes, we stained lung tissues (n ¼ 4) for expression of CD163. In healthy lung tissue, alveolar macrophages expressed little to no CD163 (Figure 3A). By contrast, monocytes/macrophages in the GPA alveolar space adjacent to the inflammatory lesions expressed large amounts of CD163 (Figures 3B and 3C). In the

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Discussion We have demonstrated that circulating monocytes in GPA patients produce less TNF-α upon stimulation compared to healthy controls, and that lesional monocytes/ macrophages in granulomatous inflammation strongly express CD163, a marker of the alternatively activated macrophages with anti-inflammatory and wound-healing properties (15). Circulating proinflammatory monocytes, characterized by high TNF-α production and the surface expression of CD16, are enriched in infectious and autoimmune

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Decreased TNF-α production in GPA

diseases (7, 19). In our study, CD16þ cells were found to be enriched in patients with GPA. This is consistent with the previous observations that the levels of activated monocytes that express CD11b and CD64 were elevated in both active and quiescent GPA (20). This raises the question of whether CD16 expression is a specific marker of the proinflammatory state or a general marker of activation and differentiation of monocytes in GPA. Indeed, Chiu et al showed that cytokines, including M-CSF, a crucial cytokine in the alternative activation of monocytes, can induce monocytes to express CD16 and that CD16þ monocytes can differentiate into osteoclasts, which are specialized MNGs with bone resorptive capacity (16). In the present study, CD16þ and CD16– monocytes in GPA and controls did not differ in TNF-α production, suggesting that CD16þ monocytes might not be more pro-inflammatory than CD16– monocytes in GPA. Although the functional role of CD16þ monocytes in GPA pathogenesis remains to be defined, it is tempting to speculate that CD16þ monocytes contribute to granulomatous inflammation by increased MNG formation and by regulating other proinflammatory cells including Thelper 17 cells, which are expanded in GPA independent of disease activity (7, 21). The decreased TNF-α production of monocytes in GPA compared with healthy controls is surprising and contrasts with previous findings in systemic lupus erythematosus (SLE). Sule et al demonstrated that monocytes in patients with SLE produced more TNF-α after activation with apoptotic cells, independent of disease activity (22). One possible explanation for the decreased TNF-α production in GPA may be the phenomenon known as LPS tolerance, in which activated monocytes produce fewer proinflammatory cytokines after repetitive LPS stimulation due to exhaustion (23). It is possible that circulating monocytes are already activated in GPA, expressing higher levels of CD16, and are therefore less responsive to in vitro LPS stimulation. Another possible explanation comes from our previous observation that circulating GPA monocytes had a higher propensity to form MNGs compared to control monocytes and this MNG formation did not correlate with disease activity or duration, but with the extent of systemic manifestation (3). Therefore, monocytes in GPA might be alternatively activated, exhibiting an increased propensity to form granulomatous inflammation at the expense of lower TNF-α production. Indeed, cytokines such as IL-4 and M-CSF can promote both MNG formation and alternative activation of monocytes (15). Of note, granulomatous inflammation is not a feature of SLE. The abundance of CD163-expressing cells indirectly suggests that monocytes/macrophages in the granulomatous inflammation of GPA lung produce less TNF-α in situ, as TNF-α production correlates indirectly with CD163 expression (data not shown). Instead, monocytes may preferentially differentiate into MNGs, contributing to the granulomatous inflammation in GPA. Similarly,

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a strong CD163 expression was observed in cells forming granuloma in sarcoidosis, a systemic granulomatous disease of unclear aetiology (24). It is possibly that impaired cellular immunity with decreased TNF-α production may contribute to the higher rate of bacterial colonization of sinonasal inflammation in GPA as well (11, 12). Our study has some limitations. The functional studies were performed with monocytes from a relatively small number of GPA patients who were receiving immunosuppressive therapy. As such, the decreased TNF production could be due to effects of the immunosuppressive treatment. Further studies are needed to investigate whether monocytes from untreated GPA patients with quiescent and high disease activity exhibit decreased TNF-α production. In addition, we were not able to demonstrate directly the decreased TNF-α production by tissue-infiltrating monocytes/macrophages in vivo because of limited accessibility to fresh GPA lung samples. In conclusion, GPA monocytes produce less TNF-α upon activation and express CD163 as a possible result of alternative activation. Understanding the mechanisms regulating monocyte polarization and differentiation might provide new insights into the pathogenesis of GPA and novel therapeutic opportunities. Acknowledgements We thank Antony Rosen and Maximilian F Konig at The Johns Hopkins University for their insightful comments, which contributed significantly to improving this manuscript.

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408 10. Wegener’s Granulomatosis Etanercept Trial (WGET) Research Group. Etanercept plus standard therapy for Wegener’s granulomatosis. N Engl J Med 2005;352:351–61. 11. Laudien M, Gadola SD, Podschun R, Hedderich J, Paulsen J, Reinhold-Keller E, et al. Nasal carriage of Staphylococcus aureus and endonasal activity in Wegener s granulomatosis as compared to rheumatoid arthritis and chronic rhinosinusitis with nasal polyps. Clin Exp Rheumatol 2010;28:51–5. 12. Stegeman CA, Tervaert JW, Sluiter WJ, Manson WL, de Jong PE, Kallenberg CG. Association of chronic nasal carriage of Staphylococcus aureus and higher relapse rates in Wegener granulomatosis. Ann Intern Med 1994;120:12–17. 13. Krysko O, Holtappels G, Zhang N, Kubica M, Deswarte K, Derycke L, et al. Alternatively activated macrophages and impaired phagocytosis of S. aureus in chronic rhinosinusitis. Allergy 2011;66:396–403. 14. Moestrup SK, Moller HJ. CD163: a regulated hemoglobin scavenger receptor with a role in the anti-inflammatory response. Ann Med 2004;36:347–54. 15. Gordon S, Martinez FO. Alternative activation of macrophages: mechanism and functions. Immunity 2010;32:593–604. 16. Chiu YG, Shao T, Feng C, Mensah KA, Thullen M, Schwarz EM, et al. CD16 (FcRgammaIII) as a potential marker of osteoclast precursors in psoriatic arthritis. Arthritis Res Ther 2010;12:R14. 17. Gordon S. Alternative activation of macrophages. Nat Rev Immunol 2003;3:23–35.

JK Park et al 18. Cheadle C, Berger AE, Andrade F, James R, Johnson K, Watkins T, et al. Transcription of proteinase 3 and related myelopoiesis genes in peripheral blood mononuclear cells of patients with active Wegener’s granulomatosis. Arthritis Rheum 2010;62:1744–54. 19. Fingerle G, Pforte A, Passlick B, Blumenstein M, Strobel M, Ziegler-Heitbrock HW. The novel subset of CD14þ/CD16þ blood monocytes is expanded in sepsis patients. Blood 1993;82:3170–6. 20. Muller Kobold AC, Kallenberg CG, Tervaert JW. Monocyte activation in patients with Wegener’s granulomatosis. Ann Rheum Dis 1999;58:237–45. 21. Wilde B, Thewissen M, Damoiseaux J, Hilhorst M, van Paassen P, Witzke O, et al. Th17 expansion in granulomatosis with polyangiitis (Wegener’s): the role of disease activity, immune regulation and therapy. Arthritis Res Ther 2012;14:R227. 22. Sule S, Rosen A, Petri M, Akhter E, Andrade F. Abnormal production of pro- and anti-inflammatory cytokines by lupus monocytes in response to apoptotic cells. PLoS One 2011;6:e17495. 23. Pena OM, Pistolic J, Raj D, Fjell CD, Hancock RE. Endotoxin tolerance represents a distinctive state of alternative polarization (M2) in human mononuclear cells. J Immunol 2011;186:7243–54. 24. Abdullah M, Kahler D, Vock C, Reiling N, Kugler C, Dromann D, et al. Pulmonary haptoglobin and CD163 are functional immunoregulatory elements in the human lung. Respiration 2012;83:61–73.

Supporting Information Additional Supporting Information may be found in the online version of this article. Supplementary Table S1: Study population. Please note: the editors are not responsible for the content or functionality of any supporting material supplied by the authors. Any queries should be directed to the corresponding author.

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Decreased tumour necrosis factor-α production by monocytes of granulomatosis with polyangiitis.

We hypothesized that monocytes in patients with granulomatosis with polyangiitis (GPA) are polarized towards alternative activation with decreased tum...
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