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Antibody-Mediated Inflammation Influx and Subsequent Pathology In

Antibody-Mediated Inflammation Influx and Subsequent Pathology In

IL-4 Is an Endogenous Inhibitor of Influx and Subsequent Pathology in Acute -Mediated

This information is current as Sohail Saleem, Zhenhua Dai, Sandra N. Coelho, Bogumila T. of October 3, 2021. Konieczny, Karel J. M. Assmann, Fady K. Baddoura and Fadi G. Lakkis J Immunol 1998; 160:979-984; ; http://www.jimmunol.org/content/160/2/979 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. IL-4 Is an Endogenous Inhibitor of Neutrophil Influx and Subsequent Pathology in Acute Antibody-Mediated Inflammation1

Sohail Saleem,* Zhenhua Dai,* Sandra N. Coelho,† Bogumila T. Konieczny,* Karel J. M. Assmann,‡ Fady K. Baddoura,§ and Fadi G. Lakkis2*

IL-4 is an immunoregulatory that has in vitro and in vivo anti-inflammatory actions. In this study we investigated whether endogenously produced IL-4 modulates inflammatory processes that occur after Abs bind to target tissue by comparing (the severity of glomerulonephritis induced by heterologous anti-glomerular basement membrane Abs in wild-type (IL-4؉/؉ ,mice to that of glomerulonephritis induced in homozygous IL-4 knockout (IL-4؊/؊) mice. Two hours after Ab injection ؊/؊

IL-4 mice had significantly higher intrarenal intercellular adhesion molecule-1 mRNA expression and intraglomerular neu- Downloaded from trophil accumulation than the IL-4؉/؉ group. Treatment of IL-4؊/؊ mice with recombinant murine IL-4 at the time of disease .induction reduced intercellular adhesion molecule-1 expression and neutrophil influx to levels observed in IL-4؉/؉ kidneys -Four days after Ab administration, untreated IL-4؊/؊ mice developed significantly greater urinary excretion, intracap illary fibrinogen deposits, and glomerular hypercellularity than IL-4؉/؉ mice. These results demonstrate that endogenous IL-4 suppresses neutrophil influx and limits tissue damage in Ab-induced glomerulonephritis, suggesting that IL-4 is an important

regulator of acute inflammatory processes. The Journal of Immunology, 1998, 160: 979–984. http://www.jimmunol.org/

L-4 is an immunoregulatory cytokine produced by Th2 lym- press greater amounts of IL-4 are less susceptible (17, 18). San- phocytes, mast cells, , and a subset of NK cells (1, tiago et al. demonstrated that IL-4 overexpression in -prone I 2). Although IL-4 induces IgE synthesis and promotes im- mice prevents glomerulonephritis by down-regulating Th1-driven mediate-type reactions, there is evidence to sug- production of complement-fixing, nephrotoxic subclasses of IgG gest that IL-4 has in vitro and in vivo anti-inflammatory actions (1, (19). It is not known, however, whether IL-4 mod- 2). IL-4 inhibits IFN-␥ production (3, 4) and suppresses IFN-␥- ulates inflammatory processes that occur after Abs deposit in renal mediated activation (5, 6). IL-4 also induces differ- tissue. These processes include leukocyte migration and activation, entiation of naive T cells to Th2 (7, 8) that secrete proteinuria, fibrin accumulation, and, eventually, glomerular by guest on October 3, 2021 additional macrophage-inhibiting such as IL-10 and fibrosis (16). IL-13 (9, 10). Other anti-inflammatory actions of IL-4 include sup- In this study we examined whether IL-4 is an endogenous in- pressing IL-8 production by (11) and reducing proco- hibitor of acute, Ab-mediated tissue inflammation by comparing agulant activity expression by activated endothelial cells (12). In the severity of anti-glomerular basement membrane (anti-GBM)3 vivo administration of IL-4 has been shown to protect against ex- nephritis in IL-4ϩ/ϩ to that in IL-4Ϫ/Ϫ mice. Murine anti- perimental arthritis and -induced lung inflamma- glomerular basement membrane (anti-GBM)3 nephritis is initiated tion in (13, 14). Pancreatic overexpression of IL-4 also pre- by binding of heterologous (, , or ) Abs of the IgG vented insulitis in nonobese diabetic mice (15). class to GBM followed by rapid neutrophil accumulation in Glomerular inflammation in humans and experimental animals glomerular tissue (20, 21). Marked proteinuria, fibrinogen depos- is often initiated by binding of Abs to target Ags in the kidney (16). its, glomerular capillary obliteration, and increased glomerular cel- The severity of Ab-mediated glomerulonephritis in rats and lupus- lularity develop within several hours to a few days after Ab bind- prone mice was found to correlate with the host’s propensity to ing (20, 21). Glomerular injury in this disease model is neutrophil mount a Th1-type characterized by predominant dependent (22–24). The present report provides direct evidence IFN-␥ and IL-2 expression (17, 18). In contrast, animals that ex- that IL-4 is an endogenous inhibitor of neutrophil infiltration and subsequent glomerular pathology in Ab-induced nephritis.

*Renal Division, Emory University School of Medicine and Veterans Affairs Med- ical Center, Atlanta, GA 30033; †Renal Division, Center of Health , Materials and Methods ‡ Federal University of Pernambuco, Recife, Brazil; Department of Pathology, Animals University of Nijmegen, Nijmegen, The Netherlands; and §Department of Pa- thology and Laboratory Medicine, Veterans Affairs Medical Center and State Six- to eight-week-old male C57BL/6 wild-type (IL-4ϩ/ϩ) mice were pur- University of New York, Buffalo, NY 14215 chased from The Jackson Laboratory (Bar Harbor, ME). C57BL/6 mice Received for publication June 11, 1997. Accepted for publication October homozygous for the disrupted IL-4 (IL-4Ϫ/Ϫ) were provided by Dr. 3, 1997. Manfred Kopf (Basel Institute of Immunology, Basel, Switzerland) (7) and The costs of publication of this article were defrayed in part by the payment of were bred at the Atlanta Veterans Affairs Medical Center animal facility. page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 3 Abbreviations used in this paper: GBM, glomerular basement membrane; NTS, 1 This work was supported by a Veterans Affairs Merit Review grant (to F.G.L.). nephrotoxic ; NRS, nonimmune rabbit serum; Up/UCr, ratio of urinary pro- 2 Address correspondence and reprint requests to Dr. Fadi G. Lakkis, Emory tein to creatinine concentration; PAS, periodic acid-Schiff; CAE, chloroacetate University and Veterans Affairs Medical Center, Research 151N, 1670 Clairmont esterase; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehy- Rd., Atlanta, GA 30033. E-mail: [email protected] drogenase; ICAM-1, intercellular adhesion molecule-1.

Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 980 ANTI-INFLAMMATORY ACTIONS OF ENDOGENOUS IL-4

Complete inactivation of IL-4 gene function was verified by performing IL-4-specific ELISA (Genzyme, Cambridge, MA) on splenocyte superna- tants collected 0, 24, 48, and 72 h after Con A (3 ␮g/ml) stimulation. Experimental protocols Expt. A. To study the effect of IL-4 on Ab-mediated neutrophil influx, anti-GBM nephritis was induced in 8- to 10-wk-old IL-4ϩ/ϩ and IL-4Ϫ/Ϫ C57BL/6 mice (n ϭ 6/group) by injecting 1 ml of heat-inactivated neph- rotoxic serum (NTS; rabbit anti-mouse GBM) i.p. (20). NTS was provided by Dr. Karel J. M. Assmann (University Hospital, Nijmegen, The Nether- lands). An additional group of IL-4Ϫ/Ϫ C57BL/6 mice (n ϭ 6) received FIGURE 1. RT-PCR analysis of intrarenal IL-4 mRNA expression be- 3000 U of recombinant murine IL-4 (a gift from Dr. Satwant Narula, fore (pre-NTS) and 2 h after nephrotoxic serum administration (post- Schering-Plough Research Institute, Kenilworth, NJ) i.v. at the time of ϩ ϩ Ϫ Ϫ ϩ ϩ Ϫ Ϫ / ϩ ϩ / Ϫ Ϫ NTS administration. Control IL-4 / and IL-4 / (n ϭ 3/group) mice NTS). Total RNA was prepared from IL-4 ( / ) and IL-4 ( / ) received 1 ml of heat-inactivated nonimmune rabbit serum (NRS; Sigma kidneys, reverse transcribed, and subjected to 38 cycles of PCR. Fif- Chemical Co., St. Louis, MO) i.p. All mice were killed 2 h later, and renal teen percent of each PCR reaction was electrophoresed on 2% agarose tissue was saved for analysis. gels and stained with ethidium bromide. Results were similar in three Expt. B. To study the effects of IL-4 on glomerular pathology that occurs separate animal experiments. L, DNA ladder (␾X174RF DNA/HaeIII subsequent to neutrophil infiltration, anti-GBM nephritis was induced in 8- fragments); C , control PCR reaction without reverse transcriptase; C , ϩ/ϩ Ϫ/Ϫ ϭ 1 2 to 10-wk-old IL-4 and IL-4 C57BL/6 mice (n 10/group) by in- control PCR reaction without cDNA. jecting 1 ml of heat-inactivated NTS i.p. on day 0 (20). Control IL-4ϩ/ϩ and IL-4Ϫ/Ϫ mice (n ϭ 3/group) received 1 ml of heat-inactivated NRS (Sigma) i.p. on day 0. Twenty-four-hour was collected on days 1 and Downloaded from 4 from individual metabolic cages (Nalgene, Inc., Rochester, NY). All New Haven, CT). Ten percent of cDNA was then subjected to 38 cycles of mice were killed at the end of the fourth day following disease induction, PCR amplification (total volume of reaction, 100 ␮l) in a Perkin-Elmer and renal tissue was saved for analysis. The urinary protein concentration Thermocycler 480 (Perkin-Elmer, Foster City, CA) using mouse IL-4-spe- was quantitated by the Bradford method (Bio-Rad Laboratories, Melville, cific primers. RT-PCR controls included “no RNA” (blank) and “no re- NY). The urinary creatinine concentration was quantitated by the picric verse transcriptase” reactions. Fifteen microliters of each RT-PCR reaction acid colorimetric method (Sigma). The ratio of urinary protein to creatinine was electrophoresed on 2% SeaKem LE agarose (American Bioanalytical, concentration (Up/UCr) was used as a measure of proteinuria (25). Natick, MA) gels and stained with ethidium bromide. RT-PCR of a - http://www.jimmunol.org/ keeping gene (HPRT) was performed to verify equal RNA and cDNA loading in the RT and PCR reactions, respectively. Kidney sections from Expt. A and B were fixed in B5 solution (Great Lakes Diagnostics, Troy, MI) followed by 10% neutral-buffered formalin (Fisher Northern blotting Scientific, Pittsburgh, PA) and embedded in paraffin wax. Hematoxylin and Total kidney RNA (20 ␮g/sample) was electrophoresed on 1% formalde- eosin, periodic acid-Schiff (PAS), Masson trichrome, and silver staining ϩ hyde-agarose gels, transferred to Hybond-N membranes (Amersham Life were performed on 4-␮m sections. Light microscopic examination was Sciences, Arlington Heights, IL), and covalently linked by UV irradiation performed in a blinded fashion by a pathologist (F.K.B.). Glomerular pa- (Stratagene, La Jolla, CA). Radiolabeled probe was prepared using 50 ng thology was assessed by examining at least 100 glomeruli/animal. The of full-length murine intercellular adhesion molecule-1 (ICAM-1) cDNA extent of glomerular hypercellularity is reported as the average number of (American Type Culture Collection, Rockville, MD) in a standard oligo- by guest on October 3, 2021 cells per glomerulus. labeling protocol (Pharmacia Biotech, Piscataway, NJ) using [32P]dCTP Neutrophil enumeration (Amersham). Approximately 1 to 2 ϫ 106 cpm of labeled probe (sp. act. ϭ ϳ1 ϫ 109 dpm/␮g DNA) were used per milliliter of hybridization solution. Kidney sections from Expt. A were fixed in 10% neutral-buffered formalin Hybridizations were performed at 42°C for 16 h, following which mem- (Fisher Scientific, Fairlawn, NJ) and embedded in paraffin wax. For neu- branes were washed at a final stringency of 0.1ϫ SSC/0.1% SDS at 55°C. trophil detection, sections were deparaffinized and stained cytochemically Autoradiography was performed using Hyperfilm-MP (Amersham) and an with the esterase substrate, naphthol AS-D chloroacetate (CAE; Sigma) intensifying screen at Ϫ70°C for 12 h. Membranes were then stripped and (26). All sections were counterstained with hematoxylin. Positive cells reprobed with GAPDH radiolabeled cDNA according to the above proce- within 100 randomly selected glomeruli per animal were enumerated by dure. Densitometric analysis of Northern blots was performed using a Mo- light in a blinded fashion (25). Intraglomerular infiltration was lecular Dynamics densitometer (Sunnyvale, CA). quantitated by calculating the average number of CAEϩ cells per glomerulus. Statistical analyses Immunofluorescence Analysis of variance (Fisher’s protected least significant difference and/or Scheffe’s test) or unpaired t test was used to compare differences between Fresh kidney tissue from Expt. A and B was mounted in OCT compound groups. The confidence interval was set at 95%. (Miles Diagnostics, Elkhart, IN) and frozen in liquid isopentane cooled on dry ice, and 4-␮m cryostat sections were prepared (Leica, Nussloch, Ger- many). Direct immunofluorescence studies were performed by fixing fro- Results zen sections in acetone followed by blocking with 10% normal goat serum Intrarenal IL-4 mRNA expression in murine anti-GBM and addition of FITC-conjugated sheep Ab directed against rabbit IgG nephritis (Zymed, South San Francisco, CA), FITC-conjugated rabbit Ab directed Ϫ/Ϫ against human fibrinogen (Dako, Copenhagen, Denmark), or FITC-conju- Complete inactivation of IL-4 gene function in IL-4 mice was gated anti-mouse CD11b (Harlan/Serotec, Indianapolis, IN) at a 1/100 verified by measuring IL-4 protein concentration in supernatants of dilution. After washing with PBS and 1% Tween-20, sections were studied Con A-stimulated splenocytes. While IL-4ϩ/ϩ splenocytes pro- under a fluorescence by a pathologist (F.K.B.) in a blinded duced up to 32 pg/ml of the cytokine over 48 h, IL-4 was not fashion. The average number of intracapillary fibrinogen deposits and in- Ͻ Ϫ/Ϫ traglomerular CD11bϩ cells was determined by examining 100 detected ( 5 pg/ml) in IL-4 supernatants. We then studied glomeruli/animal (20). intrarenal IL-4 mRNA expression before and after NTS injection. As shown in Figure 1, IL-4 mRNA measured by RT-PCR was RNA isolation and RT-PCR ϩ ϩ present in normal IL-4 / renal tissue and increased significantly Kidney tissue obtained in Expt. A was snap-frozen in liquid nitrogen and 2 h following induction of nephritis. This is consistent with our stored at Ϫ80°C. Total kidney RNA was extracted in guanidinium salt previous observation that intrarenal IL-4 mRNA and IL-4 protein solution and purified by the cesium chloride method (27). RNA samples Ͻ are up-regulated in rat anti-GBM nephritis (25). On the other hand, with OD260/280 1.6 were rejected. Five micrograms of total RNA was Ϫ/Ϫ reverse transcribed using oligo(dT) primers and Superscript reverse tran- IL-4 mice failed to express IL-4 mRNA in their kidneys in scriptase according to the manufacturer’s instructions (Life Technologies, either the basal state or after induction of nephritis (Fig. 1). The Journal of Immunology 981

FIGURE 3. Northern analysis of intrarenal ICAM-1 mRNA ex- pression before (pre-NTS) and 2 h after nephrotoxic serum adminis- tration (postNTS). A, Total RNA was prepared from kidneys of IL-4ϩ/ϩ Ϫ Ϫ Ϫ Ϫ FIGURE 2. Average intraglomerular neutrophil counts before (pre- (ϩ/ϩ), IL-4 / (Ϫ/Ϫ), or IL-4 / mice treated with recombinant mu- NTS) and 2 h after nephrotoxic serum administration (post-NTS) in rine IL-4 at the time of NTS injection (Ϫ/Ϫ*). One percent formalde- IL-4ϩ/ϩ (f), IL-4Ϫ/Ϫ (ᮀ), and IL-4Ϫ/Ϫ mice treated with recombinant hyde-agarose RNA gels from three separate animal experiments were murine IL-4 at the time of NTS injection ( ). Neutrophils were iden- probed with 32P-labeled murine ICAM-1 cDNA. One representative 32 tified by CAE histochemical staining, and the average number of neu- gel reprobed with P-labeled murine GAPDH cDNA is also shown. B, Downloaded from trophils per glomerulus (mean Ϯ SD) was determined by inspecting Densitometric analysis of intrarenal ICAM-1 mRNA expression; IL- ϩ ϩ Ϫ Ϫ Ϫ Ϫ 100 glomeruli in each kidney sample. * indicates significantly greater 4 / (f), IL-4 / (ᮀ), and IL-4 / mice treated with recombinant mu- than value in either IL-4ϩ/ϩ or IL-4-treated IL-4Ϫ/Ϫ mice ( p Ͻ 0.05). rine IL-4 at the time of NTS injection ( ) are shown. The fold increase over baseline (mean Ϯ SD) was determined by dividing the post-NTS ICAM-1 band density by the pre-NTS band density after correcting for GAPDH expression in each animal experiment. ‡, Significantly greater ϩ/ϩ Ϫ/Ϫ

Ͻ http://www.jimmunol.org/ IL-4 is an endogenous suppressor of neutrophil infiltration in than value in either IL-4 or IL-4-treated IL-4 mice ( p 0.05). murine anti-GBM nephritis The acute inflammatory response in anti-GBM nephritis is char- acterized by intraglomerular neutrophil accumulation that peaks Ϫ/Ϫ 2 h after disease induction (20). To determine whether IL-4 is an Increased glomerular pathology in IL-4 mice after endogenous regulator of this response, intraglomerular neutrophil induction of murine anti-GBM nephritis (CAEϩ cells) counts in IL-4ϩ/ϩ and IL-4Ϫ/Ϫ mice were compared Functional and morphologic renal abnormalities in murine anti- 2 h after NTS injection. As shown in Figure 2, NTS induced sig- GBM nephritis develop within several hours to a few days after Ϫ/Ϫ nificantly greater neutrophil infiltration in IL-4 (mean Ϯ SE ϭ neutrophil influx into glomerular tissue (20, 21). These abnormal- by guest on October 3, 2021 ϩ ϩ ϩ 1.7 Ϯ 0.4 CAE /glomerulus) than in IL-4 / glomeruli ities are neutrophil dependent and include proteinuria, increased ϩ (0.5 Ϯ 0.2 CAE cell/glomerulus; p Ͻ 0.05). Administration of glomerular cellularity, and fibrinogen deposits within glomerular recombinant murine IL-4 at the time of NTS injection reduced capillaries and mesangium (22–24). We, therefore, investigated Ϫ/Ϫ intraglomerular neutrophil accumulation in IL-4 kidneys to whether the glomerular lesions of anti-GBM nephritis are exag- levels comparable to those in the IL-4ϩ/ϩ group (0.5 Ϯ 0.1 and ϩ gerated in the absence of IL-4. There were no deaths following Ϯ ϩ ϩ Ϫ Ϫ 0.5 0.2 CAE cell/glomerulus, respectively; Fig. 2). Neutrophil NTS injection. Average body weights in the IL-4 / and IL-4 / ϩ/ϩ counts before NTS injection (pre-NTS) were similar in IL-4 groups were similar at the end of the experiment (27 Ϯ 3 and 28 Ϯ and IL-4Ϫ/Ϫ glomeruli (0.05 Ϯ 0.02 and 0.08 Ϯ 0.02 CAEϩ cell/ ϩ/ϩ Ϫ/Ϫ 2 g, respectively). Intense linear staining for rabbit IgG could be glomerulus, respectively; Fig. 2). Control IL-4 and IL-4 demonstrated along the GBM after NTS administration and did not mice injected with NRS did not display significant neutrophil ac- ϩ ϩ Ϫ Ϫ differ between IL-4 / and IL-4 / glomeruli (data not shown). cumulation in their glomeruli. Circulating mouse anti-rabbit IgG or intrarenal mouse IgG depos- IL-4 is an endogenous suppressor of ICAM-1 up-regulation in its were not detected. Increased protein excretion was not observed murine anti-GBM nephritis in either group at 2 h. By the fourth day of nephritis, however, IL-4Ϫ/Ϫ mice displayed worse glomerular injury than IL-4ϩ/ϩ To investigate the mechanism by which IL-4 inhibits neutrophil mice, as measured by the following parameters. migration, we studied intrarenal ICAM-1 mRNA expression be- Proteinuria (U /U ). Urinary protein excretion in the IL-4Ϫ/Ϫ fore and 2 h after NTS administration. ICAM-1 plays an important p Cr group on days 1 and 4 after NTS injection was higher than that in role in neutrophil adherence to the vascular endothelium (28). As ϩ/ϩ shown in three separate animal experiments (Fig. 3A), ICAM-1 day-matched IL-4 mice (Fig. 4). The difference in Up/UCr ratio on day 4 between the IL-4Ϫ/Ϫ group (mean Ϯ SE ϭ 71 Ϯ 6; n ϭ mRNA up-regulation following induction of nephritis was signif- ϩ ϩ Ϫ Ϫ ϩ ϩ / Ϯ ϭ icantly greater in IL-4 / than in IL-4 / kidneys. Quantitative 10) and the IL-4 group (41 7; n 10) reached statistical Ͻ densitometric analysis of ICAM-1 mRNA revealed a 3.1 Ϯ 0.3- significance ( p 0.05; Fig. 4). Administration of recombinant Ϫ/Ϫ fold increase over baseline in IL-4ϩ/ϩ kidneys compared with a murine IL-4 (1000 U i.p., twice per day) to IL-4 mice starting Ϫ/Ϫ 5.7 Ϯ 0.9-fold increase in the IL-4 group (mean Ϯ SD; p Ͻ at the time of NTS injection reduced the day 4 Up/UCr ratio to 0.05; Fig. 3B). IL-4 administration at the time of NTS injection 50 Ϯ 10 (n ϭ 4), which was not significantly different from that in ϩ ϩ reduced intrarenal up-regulation of ICAM-1 mRNA in IL-4Ϫ/Ϫ the IL-4 / group. Urinary protein excretion did not increase over mice to levels observed in IL-4ϩ/ϩ mice (3.2 Ϯ 0.4- vs 3.1 Ϯ baseline in control mice injected with NRS. 0.3-fold increase, respectively; Fig. 3, A and B). Baseline (pre- Histopathologic changes. Four days following NTS injection, the NTS) expression of ICAM-1 did not differ significantly between majority of IL-4Ϫ/Ϫ mice (8 of 10) developed diffuse renal pathol- the IL-4ϩ/ϩ and IL-4Ϫ/Ϫ groups (Fig. 3A). ogy characterized by glomerular hypercellularity and narrowing or 982 ANTI-INFLAMMATORY ACTIONS OF ENDOGENOUS IL-4

Ϯ FIGURE 5. Glomerular histopathology 4 days after induction of ne- FIGURE 4. Average proteinuria (mean SE), represented by the Up/ ϩ/ϩ Ϫ/Ϫ ϩ ϩ Ϫ Ϫ phritis in IL-4 (A) and IL-4 (B) mice. PAS-stained tissue sections U , in IL-4 / (ⅷ; n ϭ 10) and IL-4 / (⅜; n ϭ 10) mice on days 0, Cr are shown (ϫ400 magnification). Arrows point to intracapillary PASϩ Downloaded from 1, and 4 after injection of nephrotoxic serum (postNTS). * indicates Ϫ/Ϫ ϩ ϩ deposits, which are prominent in IL-4 glomeruli. These deposits significantly greater than value in day-matched IL-4 / mice ( p Ͻ were identified as fibrinogen by immunofluorescence. 0.05).

Table I. Glomerular in IL-4ϩ/ϩ and IL-4Ϫ/Ϫ http://www.jimmunol.org/ pre-NTS Four days post-NTS

IL-4ϩ/ϩ IL-4Ϫ/Ϫ IL-4ϩ/ϩ IL-4Ϫ/Ϫ (n ϭ 3) (n ϭ 3) (n ϭ 10) (n ϭ 10)

Total cells/glomerulusa 24 Ϯ 324Ϯ 227Ϯ 534Ϯ 4b Neutrophils/glomerulusa 0.04 Ϯ 0.01 0.08 Ϯ 0.01 0.15 Ϯ 0.08 0.47 Ϯ 0.23b Fibrinogen deposits/glomerulusa ND ND 1.3 Ϯ 1.1 3.2 Ϯ 1.1c a One hundred glomeruli per animal were examined in a blinded fashion. Total glomerular cellularity (mean Ϯ SD) was determined by examining PAS-stained, fixed kidney sections. Intracapillary fibrinogen deposits (mean Ϯ SD) were enumerated in frozen kidney sections stained with FITC-conjugated anti-fibrinogen Ab. by guest on October 3, 2021 b p Ͻ 0.05 compared to pre-NTS groups and post-NTS IL-4ϩ/ϩ group. c p Ͻ 0.05 compared to post-NTS IL-4ϩ/ϩ group.

obliteration of capillary lumina due to intracapillary PASϩ depos- pressor of intrarenal ICAM-1 mRNA up-regulation, neutrophil in- its (Fig. 5B). Glomerular enlargement, glomerular neutrophil in- flux, and subsequent glomerular pathology in murine anti-GBM filtrate, intramesangial PASϩ deposits, and intratubular proteina- nephritis. Previous reports have shown that IL-4 protects against cious casts were also observed on light microscopic examination. glomerulonephritis in lupus-prone mice by down-regulating neph- ϩ ϩ On the other hand, only 3 of 10 IL-4 / mice had significant rotoxic subclasses of IgG autoantibodies (18, 19). Our results dem- Ϫ Ϫ glomerular changes that resembled those observed in the IL-4 / onstrate that IL-4 also exerts anti-inflammatory actions following ϩ ϩ group. The remaining IL-4 / mice had normal renal histology binding of nephrotoxic Abs to kidney tissue. ϩ except for focal intraglomerular PAS deposits (Fig. 5A). Average The observation that endogenous IL-4 inhibits neutrophil influx ϩ ϩ glomerular cellularity and neutrophil infiltrate in IL-4 / kidneys is consistent with known pharmacologic effects of this cytokine. 4 days after NTS administration were significantly higher than Mulligan et al. reported that intratracheal administration of rIL-4 Ϫ/Ϫ those in IL-4 kidneys (Table I). Baseline (pre-NTS) glomerular suppresses neutrophil infiltration in a murine model of immune cellularity was not different between the two groups (Table I). We complex-mediated lung injury (14). Similarly, i.v. IL-4 inhibited Ϫ/Ϫ also observed that IL-4 mice developed significantly more in- neutrophil accumulation in a murine air pouch after local applica- ϩ/ϩ tracapillary fibrinogen deposits per glomerulus than IL-4 mice tion of IL-1␤ (34). We also observed in this study that IL-4 is an (Table I). Occlusion of intraglomerular capillaries with fibrinogen endogenous inhibitor of ICAM-1 mRNA up-regulation following is a central feature of murine anti-GBM nephritis (20, 21). induction of murine anti-GBM nephritis. It is likely that intrarenal ICAM-1 mRNA correlates with ICAM-1 protein expression in this Discussion model because NTS administration has been shown to increase Neutrophil accumulation in glomerular tissue is a hallmark of ICAM-1 on glomerular endothelial cells (33, 35). Mulligan et al. acute, Ab-induced glomerulonephritis (16). Their transmigration demonstrated that exogenous IL-4 suppresses immune complex- into glomeruli is facilitated by ICAMs expressed on endothelial induced ICAM-1 up-regulation in pulmonary vessels (14). Simi- cells (29). Neutrophil depletion (30, 31) or inhibiting neutrophil larly, in vitro experiments have shown that IL-4 inhibits IL-1␤- migration by blocking their adherence to the endothelium (32, 33) induced ICAM-1 up-regulation on endothelial cells (36, 37). ameliorates experimental Ab-induced glomerulonephritis. In this Taken together, these observations suggest that modulation of study we provided direct evidence that IL-4 is an endogenous sup- ICAM-1 expression is an important mechanism by which IL-4 The Journal of Immunology 983 inhibits neutrophil influx. It is possible, however, that IL-4 sup- 3. Vercelli, D., H. H. Jabara, R. P. Lauener, and R. S. Geha. 1990. IL-4 inhibits the ␥ presses neutrophil migration by additional mechanisms. For ex- synthesis of IFN- . Immunology 144:570. 4. Tanaka, T., J. Hu-Li, R. A. Seder, B. Fazekas de St. Groth, and W. E. Paul. 1993. ample, IL-4 inhibits IL-8 production by and neutrophils 4 suppresses interleukin 2 and interferon. Proc. Natl. Acad. Sci. USA (11, 38). In vivo neutralization of IL-8 has been shown to prevent 90:5914. neutrophil influx in Ab-mediated glomerulonephritis (39). IL-4 5. Hart, P. H., G. F. Vitti, D. R. Burgess, G. A. Whitty, D. S. Piccoli, and J. A. Hamilton. 1989. Potential antiinflammatory effects of interleukin 4: sup- also inhibits leukotriene B4 production by monocytes (40). Leu- pression of human tumor factor 2. Proc. Natl. Acad. Sci. USA kotriene B4 is a potent neutrophil chemoattractant (41) and may 86:3803. 6. Donnelly, R. P., M. J. Fenton, D. S. Finbloom, and T. L. Gerrard. 1990. Differ- play a role in recruiting neutrophils to the kidney in anti-GBM ential regulation of IL-1 production in human monocytes by IFNJ. Immunology nephritis (42). 145:569. We also observed in this study that IL-4Ϫ/Ϫ mice develop worse 7. Kopf, M., G. L. Gros, M. Bachmann, M. C. Lamers, H. Bluethmann, and ϩ/ϩ G. Kohler. 1993. Disruption of the murine IL-4 gene blocks Th2 cytokine re- glomerular pathology than the IL-4 group. Four days after NTS sponses. 362:245. Ϫ/Ϫ administration, IL-4 mice had significantly greater proteinuria, 8. Shimoda, K., J. v. Deursen, M. Y. Sangster, S. R. Sarawart, R. T. Carson, glomerular cellularity, and intracapillary fibrinogen deposits. Be- R. A. Tripp, C. Chu, F. W. Quelle, T. Nosaka, D. A. A. Vignali, P. C. Doherty, G. Grosveld, W. E. Paul, and J. N. Ihle. 1996. Lack of IL-4 induced Th2 response cause neutrophils play a central role in murine anti-GBM nephritis and IgE class switching in mice with disrupted gene. Nature 380:630. Ϫ Ϫ (22–24), increased pathology in IL-4 / mice could have resulted 9. Fiorentino, D., A. Zlotnik, T. Mosmann, M. Howard, and A. O’Garra. 1991. from greater neutrophil influx into their glomeruli. We did not IL-10 inhibits cytokine production by activated . J. Immunol. 147: ϩ 3815. detect significant monocyte (CD11b cell) accumulation in either 10. de Waal Malefyt, R., C. G. Figdor, R. Huijbens, S. Mohan-Peterson, B. Bennett, ϩ ϩ Ϫ Ϫ IL-4 / or IL-4 / kidneys (less than one cell per glomerulus 4 J. Culpepper, W. Dang, G. Zurawski, and J. E. de Vries. 1993. Effects of IL-13 on phenotype, cytokine production, and cytotoxic function of human monocytes. days after NTS injection). Others have shown that T lymphocytes Downloaded from J. Immunol. 151:6370. and monocytes do not contribute significantly to glomerular pa- 11. Wertheim, W. A., S. L. Kunkel, T. J. Standiford, M. D. Burdick, F. S. Becker, thology in this model (43–45). The difference in proteinuria be- C. A. Wilke, A. R. Gilbert, and R. M. Strieter. 1993. Regulation of neutrophil- Ϫ Ϫ ϩ ϩ tween IL-4 / and IL-4 / mice on the first day of nephritis was derived IL-8: the role of , dexamethasone, and IL-4. J. Immunol. 151:2166. less marked than the difference in neutrophil accumulation be- 12. Herbert, J., P. Savi, M. Laplace, A. Lale, F. Dol, A. Dumas, C. Labit, and tween the two groups. This discrepancy may be explained by neu- A. Minty. 1993. IL-4 and IL-13 exhibit comparative abilities to reduce pyrogen- induced expression of procoagulant activity in endothelial cells and monocytes. trophil-independent mechanisms of proteinuria such as nephro- FEBS Lett. 328:268. http://www.jimmunol.org/ toxic Abs, which directly alter the glomerular filtration barrier (46, 13. Allen, J. B., H. L. Wong, G. L. Costa, M. J. Bienkowski, and S. M. Wahl. 1993. 47). We suspect that glomerular hypercellularity in IL-4Ϫ/Ϫ mice Suppression of monocyte function and differential regulation of IL-4 and IL-1ra by IL-4 contribute to resolution of experimental arthritis. J. Immunol. 151:4344. resulted in part from increased mesangial cell number because 14. Mulligan, M. S., M. L. Jones, A. A. Vaporciyan, M. Howard, and P. A. Ward. mesangial cell proliferation has been observed in experimental Ab- 1993. Protective effects of IL-4 and IL-10 against immune complex-induced lung induced glomerulonephritis (48), and IL-4 has been shown to sup- injury. J. Immunol. 151:5666. 15. Mueller, R., T. Krahl, and N. Sarvetnick. 1996. Pancreatic expression of inter- press proliferation of mesangial cells in culture (49). leukin-4 abrogates insulitis and autoimmune diabetes in nonobese diabetic Our results do not exclude that IL-4 can modulate glomerulo- (NOD) mice. J. Exp. Med. 184:1093. nephritis by down-regulating the inflammatory functions of endo- 16. Wilson, C. B. 1996. Renal response to immunologic glomerular injury. In The Kidney, Vol. 2. B. M. Brenner, ed. Saunders, Philadelphia, p. 1253. by guest on October 3, 2021 thelial cells and resident macrophages. For example, IL-4 sup- 17. Coelho, S. N., S. Saleem, B. T. Konieczny, K. R. Parekh, F. K. Baddoura, and presses procoagulant expression by endothelial cells (12) and F. G. Lakkis. 1997. Immunologic determinants of susceptibility to experimental inhibits nitric oxide production by macrophages (50). It remains to glomerulonephritis: role of cellular . Kidney Int. 51:646. 18. Takahashi, S., L. Fossati, M. Iwamoto, R. Merino, R. Motta, T. Kobayakawa, and be determined whether procoagulant activity and nitric oxide re- S. Izui. 1996. Imbalance towards Th1 predominance is associated with acceler- Ϫ Ϫ lease are increased in IL-4 / glomeruli and whether they con- ation of lupus-like autoimmune syndrome in MRL mice. J. Clin. Invest. 97:1597. tribute to intracapillary thrombosis in murine anti-GBM nephritis. 19. Santiago, M.-L., L. Fossati, C. Jacquet, W. Muller, S. Izui, and L. Reininger. ␣ 1997. Interleukin-4 protects against a genetically linked lupus-like autoimmune IL-4 also stimulates IL-1RA while inhibiting IL-1 and TNF- pro- syndrome. J. Exp. Med. 185:65. duction by monocytes (5, 51). In addition, it induces IL-1 decoy 20. Assmann, K. J. M., M. M. Tangelder, W. P. J. Lange, G. Schrijver, and receptor synthesis by neutrophils (52). Although a balance be- R. A. Koene. 1985. Anti-GBM nephritis in the mouse: severe proteinuria in the heterologous phase. Virchows Arch. Abt. A Pathol. Anat. 406:285. tween IL-1 and its antagonists may determine the outcome of some 21. Schrijver, G., K. J. M. Assmann, M. J. J. T. Bogman, J. C. M. Robben, forms of experimental glomerulonephritis (53, 54), their role in R. de Waal-Malefyt, and R. A. P. Koene. 1988. Antiglomerular basement mem- murine anti-GBM nephritis is not known. brane nephritis in the mouse: study on the role of complement in the heterologous phase. Lab. Invest. 59:484. In conclusion, we demonstrated that endogenously produced 22. Schrijver, G., J. Schalkwijk, J. C. M. Robben, K. J. M. Assmann, and IL-4 down-regulates ICAM-1 expression, neutrophil influx, and R. A. P. Koene. 1989. Antiglomerular basement membrane nephritis in beige mice: deficiency of leukocytic neutral proteinase prevents the induction of albu- subsequent pathology in murine Ab-induced glomerulonephritis. minuria in the heterlogous phase. J. Exp. Med. 169:1435. In contrast to IL-4’s role in promoting IgE-mediated immediate- 23. Schrijver, G., M. J. J. T. Bogman, K. J. M. Assmann, R. M. W. D. Waal, type hypersensitivity, these results suggest that IL-4 is an inhibitor H. C. M. Robben, H. V. Gastern, and R. A. P. Koene. 1990. Anti-GBM nephritis in the mouse: role of in the heterologous phase. Kidney Int. 38:86. of acute inflammation induced by IgG Abs. 24. Feith, G. W., K. J. M. Assmann, M. J. J. T. Bogman, A. P. M. V. Gompel, J. Schalkwijk, and R. A. P. Koene. 1993. Lack of albuminuria in the early het- erologous phase of anti-GBM nephritis in beige mice. Kidney Int. 43:824. Acknowledgments 25. Lakkis, F. G., F. K. Baddoura, E. N. Cruet, K. R. Parekh, M. 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