Inflammation-Induced IL-6 Functions As a Natural Brake On

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Inﬂammation-Induced IL-6 Functions as a Natural Brake on Macrophages and Limits GN

Michael Luig,* Malte A. Kluger,* Boeren Goerke,* Matthias Meyer,* Anna Nosko,* † ‡ † Isabell Yan, Jürgen Scheller, Hans-Willi Mittrücker, Stefan Rose-John,§ Rolf A.K. Stahl,* Ulf Panzer,* and Oliver M. Steinmetz*

*Medical Clinic III and †Immunology Institute, Hamburg University Medical Center, Hamburg, Germany; ‡Institute of Biochemistry and Molecular Biology II, Medical Faculty, Heinrich-Heine University, Düsseldorf, Germany; and §Institute of Biochemistry, Christian-Albrechts-University, Kiel, Germany

ABSTRACT IL-6 can mediate proinflammatory effects, and IL-6 receptor (IL-6R) blockade as a treatment for inflammatory diseases has entered clinical practice. However, opposing effects of IL-6 have been observed in models of GN. Although IL-6 is proinflammatory in murine lupus nephritis, protective effects have been observed for IL-6 in the nephrotoxic nephritis (NTN) model of acute crescentic GN. In light of the potential dangers of IL-6–directed treatment, we studied the mechanisms underlying the contradictory findings in GN. IL-6 can signal through the membrane-bound IL-6R, which is expressed only on hepatocytes and certain leukocytes (classic), or through the soluble IL-6R, which binds the ubiquitously expressed gp130 (alternative). Preemptive treatment of mice with anti-IL-6R or anti-IL-6 worsened NTN, whereas selective blockade of alternative IL-6 signaling by the fusion protein sgp130Fc did not. FACS analysis of mouse spleen cells revealed proinflammatory macrophages express the highest levels of IL-6Ra,andin vitro treatment with IL-6 blocked macrophage proliferation. Furthermore, proinflammatory macrophages were 2 2 expanded during inflammation in IL-6 / mice. Late application of anti-IL-6 after establishment of adaptive nephritogenic immunity was sufficient to aggravate NTN within 2.5 days, a period when macrophages are active. Finally, NTN was aggravated in mice with macrophage-specific impairment of IL-6 classic signaling, coincident with enhanced macrophage proliferation and accumulation in the kidney. Our data thus reveal a novel mechanism in which IL-6–mediated dampening of macrophage activation protects tissues from overshooting immune responses. This finding has important implications for potential IL-6–directed ther- apies and supports the careful choice of recipient patients and timing.

J Am Soc Nephrol 26: 1597–1607, 2015. doi: 10.1681/ASN.2014060620

IL-6 is a pleiotropic cytokine that is involved in hepatocytes and a number of distinct leukocyte multiple pathways such as regulation of acute phase subsets.4 Thus classic signaling can only occur on responses, cell growth and differentiation, as well these deﬁnedcelltypes.However,inadditionto as metabolic processes.1 Importantly, IL-6 has this restricted signaling pathway, an alternative emerged as a potent regulator of immune responses and inﬂammation.2,3 Given these multiple func- Received June 27, 2014. Accepted September 15, 2014. tions, it is not surprising that signaling of IL-6 is complex and can occur via two different pathways. M.L. and M.A.K. contributed equally to this work. In the so-called classic IL-6 signaling pathway, IL-6 Published online ahead of print. Publication date available at binds to the membrane-bound IL-6 receptor (IL-6R) www.jasn.org. which consists of two different subunits: the IL-6Ra Correspondence: Dr. Oliver M. Steinmetz, Division of Nephrology, chain to which IL-6 binds directly, and the signal- Medical Clinic III, Hamburg University Medical Center, Martinistrasse transducing membrane glycoprotein gp130. The IL-6Ra 52, 20246 Hamburg, Germany. Email: [email protected] chain is exclusively expressed on the surface of Copyright © 2015 by the American Society of Nephrology

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IL-6 signaling pathway exists which was termed IL-6 trans- effects of recombinant IL-6.35,36 Both the mechanisms under- signaling.5,6 Here, IL-6 forms a complex with a soluble IL-6 lying IL-6–mediated protection from nephritis and the respon- receptor a chain (sIL-6Ra) that binds to the ubiquitously sible IL-6 signaling pathway (classic versus trans) remained expressed gp130. Therefore, in contrast with classic signaling, unclear. Injury in the NTN model develops independently IL-6 trans-signaling can occur on all cells of the body. The of antibodies37 but highly depends on Th1 and Th17 cell re- complex role of IL-6 and the relative contribution of each sponses38–41 as well as macrophages.42 Therefore, it is very signaling pathway in immunologically mediated disease has likely that the observed effects were mediated by these highly been the focus of many studies from the past few years.7 In this IL-6–responsive leukocyte subpopulations. In addition to the respect, IL-6 was initially identified as a B-cell differentiation anti-inflammatory role of IL-6 in NTN, a more recent study factor.8 However, subsequent studies have also revealed ef- found protective effects of IL-6 trans-signaling in AKI.43 fects on multiple other cells of the immune system such as Regarding the situation in humans, IL-6 secretion from neutrophils (PMNs), macrophages, dendritic cells (DCs), and PBMCs correlated with renal disease activity in lupus nephritis.44 T-helper (Th) cells. The discovery of IL-6 as a key cytokine Urinary IL-6 has also been suggested as a marker for renal in- required for development of the highly proinflammatory flammation in different forms of GN. Definite proof for a path- Th17 response has especially gained much attention.9,10 To- ogenic role of IL-6 in human inflammatory renal disease is, gether with TGF-b, IL-6 initiates cellular events leading to however, still lacking. Few case reports from the literature de- programming of Th17 differentiation.11,12 Many studies in scribe successful treatment of renal disease by the IL-6R antibody Th17-mediated experimental models of immune-mediated tocilizumab,45 including one patient with pANCA-associated diseases such as encephalitis, colitis, and arthritis have proven refractory GN.46 In addition, one pilot study assessed the effects the proinflammatory effects of IL-6.13–15 As a consequence, of tocilizumab in patients with mild lupus nephritis.47 Although IL-6–directed therapy has been introduced into clinical prac- arthritis in these patients improved significantly, improvement tice. The mAb tocilizumab, which is directed against the hu- of renal function was less evident. Nevertheless, given the pos- man IL-6R, is successfully used to treat rheumatoid arthritis itive results in animal studies of SLE and the paucity of effective and Still’s disease.16–18 However, some significant caveats re- therapeutic options for human lupus and ANCA-associated ne- main. Besides its proinflammatory role, IL-6 signaling has also phritis, it would be very tempting to initiate randomized clinical been shown to exert strong anti-inflammatory effects. Studies trials of IL-6R blockade in GN. However, for such studies to be from the pre-Th17 era predominantly declared IL-6 to be an launched, it is crucial to better understand which mechanisms anti-inflammatory cytokine with IL-10–like properties.19–22 underlie the strong anti-inflammatory and tissue-protective ef- These effects include downregulation of macrophage prolifera- fects of IL-6. This study therefore aimed to (1) assess the effect of tion and activation23–25 as well as inhibition of DC maturation.26 IL-6 pathway blockade on the clinical course of NTN, (2)iden- Most importantly, IL-6R blockade in humans has also been tify the differential IL-6 signaling pathways that are involved reported to cause inflammatory eye disease,27 psoriasis,28 as (classic versus trans-signaling), and (3) identify which cell pop- well as crescentic immune complex GN.29 Generally, the anti- ulations mediate the effects of IL-6 in NTN. inflammatory actions of IL-6 stillremainvery ill defined. This is also the case regarding inflammatory renal disease where conflicting data have been reported. Several studies have been RESULTS performed in rodent models of SLE. Pioneering work by Kiberd showed an anti-inflammatory role of an IL-6R antibody in IL-6R Blockade Aggravates NTN the Mrl/lpr model of lupus nephritis.30 Much later, a study in NTN was induced in C57BL/6 mice and one group was treated IL-6–deficient mice confirmed these findings in the same with anti-IL-6R antibody,48 whereas controls were injected model.31 In a second model of SLE, three studies supported with control IgG. Analyses of kidneys at day 10 after NTN proinflammatory effects of IL-6. First, recombinant IL-6 induction revealed significantly aggravated disease in anti- aggravated pathogenic immune responses and the clinical course IL-6R–treated mice in terms of histologic and functional dam- of SLE in NZW3NZB mice.32 Application of anti–IL-6R as well age (Figure 1A). Renal leukocyte influx was significantly as anti–IL-6 antibodies were subsequently shown to ameliorate increased (Figure 1B) and renal Th1 and Th17 responses re- disease by two independent groups.33,34 Mechanistically, the del- mained intact (Figure 1C). Analyses of systemic T- and B-cell eterious effects of IL-6 were largely ascribed to production of immune responses showed multiple effects. Antigen-specific pathogenic antibodies. Some authors also suspected IL-6 effects antibody production was impaired, confirming effective on T-cell activation; however, this was not addressed any further blockade of IL-6 signaling (Supplemental Figure 1A). Spleen and effects on Th17 responses have not yet been studied. cell numbers were significantly reduced but regulatory T-cell Although these data are in line with and clearly indicate a percentages remained unchanged (Supplemental Figure 1, B proinflammatory role for IL-6 in lupus and lupus nephritis, and C). By contrast, Th cell activation and proliferation were results from another model of nephritis were contradictory. strongly downregulated by anti–IL-6R treatment (Supplemental Twostudies in the nephrotoxic nephritis (NTN) model of acute Figure 1, D and E). In line, production of various T-cell crescentic GN in the rat have documented anti-inflammatory cytokines was greatly diminished, including near absence

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Figure 1. anti-IL-6R treatment aggravates GN. (A) Representative photographs of PAS-stained kidney sections (left) and quantification of renal histologic damage (middle) and function (right) in nephritic aIL-6R– or control IgG–treated mice at day 10 after induction of NTN. (B) Representative photographs (left) and quantification (right) of glomerular and interstitial T cells (CD3) and macrophages (MAC2, F4/80). (C) Representative FACS plots and quantification of renal Th17 and Th1 subsets. Circles represent individual animals, whereas horizontal lines indicate mean values. Error bars represent the SD. Numbers in FACS plots represent percentages of gated events. *P,0.05; **P,0.01; ***P,0.001. PAS, periodic acid–Schiff. Original magnification, 3400. of splenic IL-17 production in anti-IL-6R–treated mice robust renal Th1 and Th17 responses in all experimental groups (Supplemental Figure 1F). (Figure 2C). Systemic B- and T-cell immunity was much less affected than in the aIL-6R experiments. Antigen-specificIgG Blockade of IL-6 Classic Signaling but Not Trans- production was similar between the groups (data not shown). Signaling Aggravates NTN Spleen cell numbers in aIL-6–treated mice were slightly reduced In a next step, NTN was again induced and mice were treated (Supplemental Figure 2A), but production of most cytokines with an antibody blocking IL-6 or the fusion protein sgp130Fc, produced by spleen cells remained comparable (Supplemental which specifically blocks IL-6 trans-signaling or control IgG. Figure 2B). Similar to anti-IL-6R–treated mice, nephritis was aggravated in the aIL-6 group in terms of histology and functional parameters IL-6 Reduces Macrophage Proliferation and Activation (Figure 2A). By contrast, no significant proinflammatory effect was In Vitro and In Vivo noted in sgp130Fc-treated mice. In line with histology, analyses of We next wanted to assess the effect of IL-6 on macrophages because renal leukocytes showed enhanced infiltration in aIL-6–treated anti-inflammatory effects have been postulated. FACS analyses of mice (Figure 2B). T-cell subtype characterization again revealed spleen cells indeed showed strong expression of IL-6Ra

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Figure 2. Blockade of the classic IL-6 pathway aggravates GN. (A) Representative photographs of PAS-stained kidney sections (left) and quantification of renal histologic damage (middle) and function (right) in nephritic control IgG–, sgp130Fc–, or aIL-6–treated mice at day 10 after induction of NTN. (B) Quantification of glomerular and interstitial T cells (CD3) and macrophages (MAC2, F4/80). (C) Rep- resentative FACS plots and quantification of renal Th17 and Th1 subsets. Circles represent individual animals, whereas horizontal lines indicate mean values. Error bars represent SD. *P,0.05; **P,0.01; ***P,0.001. PAS, periodic acid–Schiff. Original magnification, 3400. preferentially on inflammatory Ly6Chigh macrophages. Expres- neutrophils, which are very early mediators of NTN, anti-IL-6, sion on all other myeloid cells measured, including Ly6Cint/lo sgp130Fc, or control IgG was administered at an interval of macrophages, DCs, and neutrophils, was strikingly lower 36 hours after NTN induction. Treatment with aIL-6 resulted (Figure 3, A and B). This identified proinflammatory macro- in significant aggravation at day 7 of nephritis in terms of phages as primary targets for classic IL-6 signaling. We there- histology, function, and leukocyte influx. Again, no deleterious fore isolated peritoneal macrophages and incubated them in effects were seen in sgp130Fc-treated mice (Figure 4, A and B). the presence or absence of IL-6. Assessment of both numbers of Next, we wanted to assess whether aggravation of nephritis colony forming units and cell numbers per colony, revealed could also be observed at earlier time points after anti–IL-6 strong inhibitory effects of IL-6 on macrophage proliferation application. Therefore, in a second set of experiments, acceler- (Figure 3C). ated NTN (aNTN) was again induced by the same protocol and 2 2 To assess in vivo effects of IL-6 on macrophages, IL-6 / organs were harvested as early as day 4. Again analyses showed mice and age-matched wild-type controls were immunized significant aggravation of nephritis in the anti-IL-6–treated with non-nephritogenic sheep IgG. Two days later, macrophages group only 2.5 days after antibody treatment, pointing to effects from bone marrow, spleen, and blood were analyzed by flow on rapidly acting cells like macrophages (Figure 4C). cytometry. Although analysis of bone marrow as the internal control did not reveal differences, percentages of highly activated Macrophage Restricted Abrogation of IL-6 Signaling Ly6Chigh macrophages were significantly enhanced in spleens Induces a Proinflammatory Phenotype and Aggravates 2 2 and even more so in blood from IL6 / mice (Figure 3D). These NTN effects were specificforLy6Chigh macrophages, because percen- Our previous experiments suggested a role for classic IL-6 tages of Ly6Cint/lo macrophages, DCs and neutrophils were signaling in macrophages as the underlying mechanism for slightly reduced in blood and spleens (not shown). In addition, theobservedaggravationofnephritis.Therefore,micelacking 2 2 blood monocytes from IL6 / mice showed much higher theIL-6Ronmacrophagesbutnotonadaptiveimmunecells fl fl MHCII expression (Figure 3E). were generated by intercrossing LysMCre with IL-6Ra ox/ ox mice. Effective knockout of IL-6Ra was confirmed by anal- Blockade of IL-6 Classic Signaling in the Effector Phase ysis of IL-6Ra expression of thioglycollate-elicited peritoneal Aggravates NTN myeloid cells49 (Supplemental Figure 3). NTN induction in fl fl In order to exclude IL-6 effects on the development of adaptive LysMCre3IL-6Ra ox/ ox mice again showed significant aggravation immunity and to assess specific effects on the macrophage- compared with IL-6R intact controls in terms of histology, func- dependent effector phase of nephritis, we switched to the tional impairment, and leukocyte infiltration (Figure 5, A and B). model of accelerated NTN. Five days after preimmunization, Importantly, FACS analyses of renal myeloid cells showed signifi- NTN serum was injected. In order to minimize effects on cant skewing in favor of proinflammatory Ly6Chigh macrophages,

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Figure 3. Proinflammatory macrophages respond to IL-6. (A) Percentages of IL-6Ra–expressing cells of naïve splenic Ly6chigh and Ly6cint/low macrophages, DCs, and neutrophils (PMNs). (B) Quantification of IL-6Ra mean fluorescence intensity (MFI) in the respective populations. (C) Quantification of macrophage colony forming units (left) and cells per colony (right) with and without addition of IL-6. 2 2 (D) FACS quantification of Ly6chigh macrophages in indicated compartments of IL-6 / mice. (E) Quantification of MHCII expression on blood macrophages. Circles represent individual animals, whereas horizontal lines indicate mean values. Numbers in FACS plots represent percentages of gated events. ***P,0.001. whereas percentages of infiltrating neutrophils remained unchanged DISCUSSION (Figure5C).Inaddition,proliferationratesoftotalmacrophagesas well as of the Ly6Chigh proinflammatory subset were enhanced in Our study aimed to evaluate the therapeutic potential of IL-6 fl fl kidneys of LysMCre3IL-6Ra ox/ ox mice (Figure 5D). By contrast, blocking strategies in crescentic GN. Our initial hypothesis was percentages of mannose receptor-positive anti-inflammatory M2 that interference with IL-6 signaling would ameliorate disease macrophages were greatly reduced (Figure 5E). DC percentages by impairing generation of Th17 responses, which have been and proliferation remained unchanged (Supplemental Figure 4A). shown to be central mediators of GN.40,50 Antibody blockade As expected, analyses of renal Th cells showed unaltered balances of the IL-6R, however, surprisingly resulted in disease aggra- of Th1 and Th17 responses as well as regulatory T-cells (Sup- vation. As expected, antigen-specific antibody and systemic plemental Figure 4B). Systemic immunity including splenocyte cytokine production were greatly reduced, indicating the potency composition, T-cell activation, and cytokine production was also of aIL-6R treatment. Th17 responses in the acutely inflamed kid- similar between the groups (Supplemental Figure 4, C and D). ney, however, remained intact. Various different mechanisms

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Figure 4. Blockade of classic IL-6 signaling in the effector phase aggravates GN. (A) Experimental outline (top), representative photographs of PAS-stained kidney sections (left) and quantification of renal histologic damage (middle) and function (right) in nephritic mice treated with control IgG, sgp130Fc or aIL-6 at day 7 after induction of aNTN. (B) Quantification of glomerular and interstitial T cells (CD3) and macrophages (MAC2, F4/80). (C) Experimental outline (top), representative photographs of PAS-stained kidney sections (left) and quantification of renal histologic damage (right) in nephritic control IgG–, sgp130Fc–,oraIL-6–treated mice at day 4 after induction of aNTN. Circles represent individual animals, whereas horizontal lines indicate mean values. *P,0.05; **P,0.01; ***P,0.001. PAS, periodic acid–Schiff. Original magnification, 3400. could account for this observation. First, IL-6 is needed for aggravation occurred although renal and systemic adaptive im- initiation of Th17 generation but not for their expansion so that mune responses remained largely unchanged by anti–IL-6 treat- preformed Th17 cells can proliferate in the absence of IL-6. In ment, which pointed toward a role for innate mediators. Because addition, some studies have identified so-called natural Th17 aggravation was mediated by classic signaling, the effects were cells that are already generated in the thymus and do not depend unlikely to be mediated by resident nonhematopoietic renal cells on further IL-6.51,52 Second, antibody blockade is never com- as these do not express the IL-6R.43,55 Rather, aggravation of plete and small remaining amounts of active IL-6 are probably disease was more likely to be affected by leukocytes, which, ex- sufficient to trigger Th17 differentiation, especially in a highly cept for hepatocytes, are the only cells known to express IL-6R inflammatory environment like a nephritic kidney and the cor- on their surface.56 In this regard, we wanted to study the role of responding lymph node. Finally, it has been shown that other macrophages because macrophages are known to be potent ef- factors including IL-21 can substitute for IL-6 and can indepen- fectors of tissue injury in NTN42 and IL-6 was postulated to dently induce Th17 responses.10,53 Given the multiple effects of downregulate macrophage activation and proliferation.23–25 In IL-6R blockade on a broad range of immune responses, we next line, preferentially proinflammatory Ly6Chigh macrophages ex- wanted to investigate whether aggravation of nephritis was spe- pressed the IL-6Ra. By contrast, expression levels on Ly6Cint/low cifically attributable to the anti–IL-6R antibody we used or if macrophages, DCs, and especially neutrophils were much lower. they were more general in nature. We thus applied a different Because high constitutive IL-6R expression was found on almost antibody that blocks IL-6 rather than IL-6R. Furthermore, we all Ly6chigh macrophages from naïve mice, inflammation-induced wanted to evaluate which IL-6 signaling pathway is involved in IL-6 could act as a rapid brake on these cells. Supporting this aggravation of disease and thus applied the designer protein concept, immunization led to rapidly enhanced systemic numbers sgp130Fc, which specifically blocks IL-6 trans-signaling.54 In of proinflammatory macrophages in mice genetically lacking line with our first experiment, application of anti–IL-6 aggra- IL-6. Vice versa, macrophage proliferation could be greatly re- vated nephritis. Trans-signaling blockade did not alter any of the duced by in vitro treatment with IL-6. In order to study the in examined end points, indicating that classic IL-6 signaling me- vivo role of macrophages in nephritis, we used the model of diated the observed protective effects of IL-6. Interestingly, accelerated NTN. In this model, mice are preimmunized with

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Figure 5. Abrogation of IL-6 classic signaling in macrophages aggravates GN. (A) Representative photographs of PAS-stained kidney fl fl sections (left) and quantification of renal histologic damage (middle) and function (right) in nephritic LysMCre or LysMCre3IL-6Ra ox/ ox mice at day 10 after induction of NTN. (B) Quantification of glomerular and interstitial T cells (CD3), macrophages (MAC2, F4/80), and neutrophils (GR-1). (C) Representative FACS plots (left) and quantification (right) of renal Ly6chigh and Ly6cint/low macrophage subsets as well as Ly6Ghi PMNs. (D) Quantification of renal macrophage subset proliferation. (E) Quantification of Mannose receptor/CD206 expression on renal macrophage subtypes. Circles represent individual animals, whereas horizontal lines indicate mean values. Error bars represent the SD. Numbers in FACS plots represent percentages of gated events. One representative of two experiments performed is shown. *P,0.05; **P,0.01; ***P,0.001. Original magnification, 3400. non-nephritogenic sheep IgG so that adaptive immune responses spares the very early neutrophil-dependent phase of NTN.57 can develop during the following days. After establishment of Again we found significant aggravation of NTN by anti–IL-6 adaptive immunity, NTN is induced by injection of nephritogenic but not by sgp130Fc. Importantly, aggravation of nephritis was sheep IgG, which binds to the glomerular basement membrane observed as early as 2.5 days after anti-IL-6 antibody application. and acts as a planted glomerular antigen.41 The preformed adap- This finding indicated IL-6 effects on rapid immune effectors such tive immune responses then mediate tissue damage by recruit- as macrophages. In order to provide compelling evidence that ment of macrophages as effectors. Treatment with anti–IL-6 and impaired classic IL-6 signaling in macrophages is causative for sgp130Fc was started 36 hours after injection of the nephritogenic aggravation of nephritis, we generated mice specifically lacking serum so that only the macrophage-dependent effector phase, but the membrane-bound IL-6Ra subunitonthemyeloidcelllineage not the previous development of adaptive immunity, was affected. including macrophages. In line with our hypothesis, induction of Importantly, by delaying treatment for 36 hours, this protocol also NTN indeed showed more severe disease in the knockout mice.

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Importantly, renal proinflammatory macrophage numbers and applied in doses previously shown to be effective, including anti–IL-6R proliferative activity were significantly and selectively enhanced. (750 mg, MR16-1; provided by Chugai Pharmaceutical Co. Ltd., Tokyo, Mannose receptor-positive M2 macrophages in contrast were Japan33), anti–IL-6 (500 mg, MP5-20F3), and sgp130Fc (250 mg),61 reduced, indicating a switch toward a predominance of the or control antibodies (750 mg rat IgG for MR16-1 or 500 mghIgGfor M1 type.58 Further analyses showed that percentages of renal anti–IL-6 and sgp130Fc) were intraperitoneally injected 24 hours neutrophils were similar between the groups. Likewise, DC per- before and 5 days after NTN induction. For aNTN,41 mice were centages and proliferative activity remained unchanged, as did preimmunized with sheep IgG in complete Freund’s adjuvant at T-cell activation and cytokine production. Although we cannot day 5. NTN was induced at day 0 and antibodies were administered fully rule out contributions of neutrophils and DCs to the ob- intraperitoneally 36 hours later. Organs were harvested at day 10 served aggravation of nephritis, our data clearly support a after NTN induction and day 4 or 7 after aNTN induction as indicated. key role for macrophages. It is likely that inflammation-induced Animal experiments were performed according to national and institu- IL-6 physiologically acts as a rapid downregulator of proin- tional animal care and ethical guidelines and were approved by local flammatory macrophage proliferation and an inductor of M2 committees (approval code G37/11). Urine samples were collected after polarization to protect the organism from overshooting immune housing the mice in metabolic cages. Albuminuria was determined by reactions. Interestingly, a very recent independent study published standard ELISA (Bethyl Laboratories). BUNand urinary creatinine were during preparation of this article reported the same key finding of quantified using standard laboratory methods. increased macrophage activation in the absence of IL-6Ra signaling that aggravated insulitis.59 This points toward a more general Morphologic Studies role of IL-6–mediated dampening of macrophage activation in Crescent formation and glomerular necrosis were determined in a inflammatory diseases. What implications do our observations minimum of 50 glomeruli per mouse in 2-mm–thick periodic acid– have on IL-6 blocking strategies in renal inflammatory diseases? Schiff-stained kidney sections in a blinded manner. Semiquantitative The consequences of IL-6 pathway blockade might be context analysis of tubulointerstitial damage was performed using 10 randomly dependent. Targeting IL-6 seems to be especially useful for diseases selected cortical areas (3200) as previously described.41 Paraffin- with certain characteristics, particularly those in which patho- embedded sections were stained with antibodies directed against genic IL-6–dependent autoantibodies are involved as SLE. Fur- CD3 (A0452; Dako, Hamburg, Germany), F4/80 (BM8; BMA Biomedicals, thermore, in general, diseases with a more chronic course might Hiddenhausen, Germany), MAC2 (M3/38; Cedarlane Laboratories, be favorably influenced by continuous IL-6 pathway blockade. Burlington, ON, Canada), GR-1, or Foxp3 and developed with a polymer- Conversely, however, during acute renal inflammation as in a based secondary antibody–alkaline phosphatase kit (POLAP; Zytomed, disease flare, blockade might be deleterious. Finally, because the Berlin, Germany), as previously published.60 Fifty glomerular cross- protective effects of IL-6 were mediated by classic signaling, spe- sections and 30 tubulointerstitial high-power fields (3400 magnification) cific blockade of IL-6 trans-signaling might be a promising al- per kidney section were counted in a blinded fashion. ternative, especially because sgp130Fc is already being tested in clinical phase I studies. Isolation of Leukocytes from Various Tissues In summary, our data reveal the novel finding that inflammation- Spleens were harvested in HBSS and passed through 70-mm nylon induced IL-6 acts as a rapid brake on proinflammatory macro- meshes. After lysis of erythrocytes with ammonium chloride, cells were phage responses. Thisobservationhas great implicationsbecause washed and passed over 40-mm meshes. Cells were then washed again, it might constitute a common mechanism protecting the counted, and resuspended in PBS for either culture or FACS analysis. organism from overshooting immune responses during acute Kidneys were minced and incubated in digestion-medium (RPMI 1640 inflammatory diseases. medium containing 10% FCS, 1% HEPES, 1% penicillin/streptomycin, 8 mg/ml collagenase D, and 0.4 mg/ml DNase) at 37°C for 40 minutes. Tissues were then homogenized, passed over 70-mmnylonmeshes,and CONCISE METHODS centrifuged at 300g at 4°C for 5 minutes. After lysis of erythrocytes with ammonium chloride, cells were filtered over 40-mm meshes, washed Animals again, and sedimented for 15 minutes at 4°C. The upper leukocyte frac- 2 2 IL6 / and LysMCre (B6.129P2-Lyz2tm1(cre)Ifo/J)micewerepro- tion was aspirated and filtered through another 40-mm nylon mesh. vided by Gisa Tiegs (Hamburg, Germany). IL-6Ra floxed mice Cells were washed, counted, and resuspended in PBS for staining and (B6;SJL-Il6ratm1.1Drew/J) were a kind gift from Juergen Scheller FACS analysis. Peripheral blood was drawn into EDTA-coated tubes and (Düsseldorf, Germany). All mice used were on a C57BL/6 back- red blood cell lysis was performed after staining for surface markers. ground and initially derived from the The Jackson Laboratory and Bone marrow cells were isolated from femoral and humeral bones, were bred at our animal facility. as previously described.62 In brief, bones were stored in ice-cold PBS, dissected, and flushed using a syringe filled with ice-cold PBS under Animal Experiments and Functional Studies sterile conditions. Afterward, cells were filtered through a 40-mm mesh Nephrotoxic nephritis was induced in 8- to 10-week-old male mice of and washed twice by centrifugation at 300g for 10 minutes at 4°C. the indicated genotypes by intraperitoneal injection of 2.5 mg of For analysis of peritoneal myeloid cells, mice were injected nephrotoxic sheep serum per gram of body weight.60 Antibodies were intraperitoneally with 1 ml thioglycollate. The peritoneal cavity was

1604 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 1597–1607, 2015 www.jasn.org BASIC RESEARCH washed with 5 ml ice-cold PBS to harvest cells at day 3 (for real-time Statistical Analyses quantitative PCR) or day 8 (for flow cytometry). Results are expressed as the mean6SD. Groups were compared by the t test and a P value,0.05 was considered statistically significant. In Antigen-Specific Systemic Cellular and Humoral the case of multiple comparisons, one-way ANOVAwas applied using Immune Responses Tukey post hoc testing. Splenocytes (43106 cells/ml) were cultured under standard conditions in the presence of normal sheep IgG (10 mg/ml; Sigma-Aldrich, Taufkirchen, Germany) and supernatants were harvested after 72 hours. Commercially available ELISAs were used for detection ACKNOWLEDGMENTS of IFN-g, IL-4, IL-6, TNFa, IL-10, IL-17A (Biolegend, San Diego, CA) 2/2 Cre and TGF-b (R&D Systems, Minneapolis, MN). Circulating sheep- We thank Professor Gisa Tiegs for providing IL-6 and LysM mice. globulin–specific serum IgG titers were analyzed by ELISA (total IgG; MR16-1 antibody was kindly provided by Tadamitsu Kishimoto and Biozol, Eching, Germany). Chugai Pharma (Osaka, Japan). We thank M. Schaper and M. Reszka for their excellent technical help. Flow Cytometry This work was supported by grants from the German Research Cells were surface stained for 30 minutes at 4°C with fluorochrome- Foundation (STE 1822/2-1 and KFO 228 STE 1822/3-1 to O.M.S., labeled antibodies against CD45, CD3, CD4, CD19, CD44, CD69, KFO 228 to H.-W.M., and SFB841 [project C1] and SFB877 [project CD62L, gd TCR, IL-6Ra, F4/80, Ly6C, Ly6G, CD206, and MHCII A1] to S.-R.J.). (all Ebioscience, San Diego, CA) and CD11b (BD Biosciences, Heidelberg, Germany) as previously described.60,62 After pregating on CD45+ live 2 2 cells, macrophages were gated as CD11b+Ly6G CD11c and Ly6Chigh DISCLOSURES or Ly6Cint/low. DCs were gated as CD11b+CD11c+MHCII+, and neutro- S.-R.J. is a shareholder of the CONARIS Research Institute AG (Kiel, phils were gated as CD11b+Ly6G+. For intracellular and intranuclear Germany), which is commercially developing sgp130Fc as a therapy for in- staining, samples were processed using a commercial intranuclear staining flammatory diseases, and is an inventor on gp130 patents owned by CONARIS. kit (Foxp3 Staining Kit; Ebioscience). Fluorochrome-labeled antibodies against IL-17, IFN-g, Foxp3, and Ki67 (Ebioscience) were utilized as recently published.60 For intracellular cytokine staining, cells were ac- REFERENCES tivated with PMA (50 ng/ml; Sigma-Aldrich) and ionomycin (1 mg/ml; Calbiochem-Merck) for 5 hours. After 30 minutes of incubation, brefeldin 1. Neurath MF, Finotto S: IL-6 signaling in autoimmunity, chronic inflammation and inflammation-associated cancer. Cytokine Growth A(10mg/ml; Sigma-Aldrich) was added. 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