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The Journal of Immunology, 1998, 160: 979-984.
Copyright © 1998 by The American Association of Immunologists

IL-4 Is an Endogenous Inhibitor of Neutrophil Influx and Subsequent Pathology in Acute Antibody-Mediated Inflammation1

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

* Renal Division, Emory University School of Medicine and Veterans Affairs Medical Center, Atlanta, GA 30033; {dagger} Renal Division, Center of Health Sciences, Federal University of Pernambuco, Recife, Brazil; {ddagger} Department of Pathology, University of Nijmegen, Nijmegen, The Netherlands; and § Department of Pathology and Laboratory Medicine, Veterans Affairs Medical Center and State University of New York, Buffalo, NY 14215


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IL-4 is an immunoregulatory cytokine 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 gene knockout (IL-4-/-) mice. Two hours after Ab injection, IL-4-/- mice had significantly higher intrarenal intercellular adhesion molecule-1 mRNA expression and intraglomerular neutrophil 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 protein excretion, intracapillary 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IL-4 is an immunoregulatory cytokine produced by Th2 lymphocytes, mast cells, basophils, and a subset of NK cells (1, 2). Although IL-4 induces IgE synthesis and promotes immediate-type hypersensitivity reactions, there is evidence to suggest that IL-4 has in vitro and in vivo anti-inflammatory actions (1, 2). IL-4 inhibits IFN-{gamma} production (3, 4) and suppresses IFN-{gamma}-mediated macrophage activation (5, 6). IL-4 also induces differentiation of naive T cells to Th2 lymphocytes (7, 8) that secrete additional macrophage-inhibiting cytokines such as IL-10 and IL-13 (9, 10). Other anti-inflammatory actions of IL-4 include suppressing IL-8 production by neutrophils (11) and reducing procoagulant activity expression by activated endothelial cells (12). In vivo administration of IL-4 has been shown to protect against experimental arthritis and immune complex-induced lung inflammation in rats (13, 14). Pancreatic overexpression of IL-4 also prevented insulitis in nonobese diabetic mice (15).

Glomerular inflammation in humans and experimental animals is often initiated by binding of Abs to target Ags in the kidney (16). The severity of Ab-mediated glomerulonephritis in rats and lupus-prone mice was found to correlate with the host’s propensity to mount a Th1-type immune response characterized by predominant IFN-{gamma} and IL-2 expression (17, 18). In contrast, animals that express greater amounts of IL-4 are less susceptible (17, 18). Santiago et al. demonstrated that IL-4 overexpression in lupus-prone mice prevents glomerulonephritis by down-regulating Th1-driven production of complement-fixing, nephrotoxic subclasses of IgG autoantibodies (19). It is not known, however, whether IL-4 modulates inflammatory processes that occur after Abs deposit in renal tissue. These processes include leukocyte migration and activation, proteinuria, fibrin accumulation, and, eventually, glomerular fibrosis (16).

In this study we examined whether IL-4 is an endogenous inhibitor of acute, Ab-mediated tissue inflammation by comparing the severity of anti-glomerular basement membrane (anti-GBM)3 nephritis in IL-4+/+ to that in IL-4-/- mice. Murine anti-glomerular basement membrane (anti-GBM)3 nephritis is initiated by binding of heterologous (sheep, rabbit, or goat) Abs of the IgG class to mouse GBM followed by rapid neutrophil accumulation in glomerular tissue (20, 21). Marked proteinuria, fibrinogen deposits, glomerular capillary obliteration, and increased glomerular cellularity develop within several hours to a few days after Ab binding (20, 21). Glomerular injury in this disease model is neutrophil dependent (22, 23, 24). The present report provides direct evidence that IL-4 is an endogenous inhibitor of neutrophil infiltration and subsequent glomerular pathology in Ab-induced nephritis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Six- to eight-week-old male C57BL/6 wild-type (IL-4+/+) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). C57BL/6 mice homozygous for the disrupted IL-4 genes (IL-4-/-) were provided by Dr. Manfred Kopf (Basel Institute of Immunology, Basel, Switzerland) (7) and were bred at the Atlanta Veterans Affairs Medical Center animal facility. Complete inactivation of IL-4 gene function was verified by performing IL-4-specific ELISA (Genzyme, Cambridge, MA) on splenocyte supernatants collected 0, 24, 48, and 72 h after Con A (3 µg/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-4+/+ and IL-4-/- C57BL/6 mice (n = 6/group) by injecting 1 ml of heat-inactivated nephrotoxic serum (NTS; rabbit anti-mouse GBM) i.p. (20). NTS was provided by Dr. Karel J. M. Assmann (University Hospital, Nijmegen, The Netherlands). An additional group of IL-4-/- C57BL/6 mice (n = 6) received 3000 U of recombinant murine IL-4 (a gift from Dr. Satwant Narula, Schering-Plough Research Institute, Kenilworth, NJ) i.v. at the time of NTS administration. Control IL-4+/+ and IL-4-/- (n = 3/group) mice received 1 ml of heat-inactivated nonimmune rabbit serum (NRS; Sigma Chemical Co., St. Louis, MO) i.p. All mice were killed 2 h later, and renal tissue was saved for analysis.

Expt. B. To study the effects of IL-4 on glomerular pathology that occurs subsequent to neutrophil infiltration, anti-GBM nephritis was induced in 8- to 10-wk-old IL-4+/+ and IL-4-/- C57BL/6 mice (n = 10/group) by injecting 1 ml of heat-inactivated NTS i.p. on day 0 (20). Control IL-4+/+ and IL-4-/- mice (n = 3/group) received 1 ml of heat-inactivated NRS (Sigma) i.p. on day 0. Twenty-four-hour urine was collected on days 1 and 4 from individual metabolic cages (Nalgene, Inc., Rochester, NY). All mice were killed at the end of the fourth day following disease induction, and renal tissue was saved for analysis. The urinary protein concentration was quantitated by the Bradford method (Bio-Rad Laboratories, Melville, NY). The urinary creatinine concentration was quantitated by the picric acid colorimetric method (Sigma). The ratio of urinary protein to creatinine concentration (Up/UCr) was used as a measure of proteinuria (25).

Histopathology

Kidney sections from Expt. A and B were fixed in B5 solution (Great Lakes Diagnostics, Troy, MI) followed by 10% neutral-buffered formalin (Fisher Scientific, Pittsburgh, PA) and embedded in paraffin wax. Hematoxylin and eosin, periodic acid-Schiff (PAS), Masson trichrome, and silver staining were performed on 4-µm sections. Light microscopic examination was performed in a blinded fashion by a pathologist (F.K.B.). Glomerular pathology was assessed by examining at least 100 glomeruli/animal. The extent of glomerular hypercellularity is reported as the average number of cells per glomerulus.

Neutrophil enumeration

Kidney sections from Expt. A were fixed in 10% neutral-buffered formalin (Fisher Scientific, Fairlawn, NJ) and embedded in paraffin wax. For neutrophil detection, sections were deparaffinized and stained cytochemically with the esterase substrate, naphthol AS-D chloroacetate (CAE; Sigma) (26). All sections were counterstained with hematoxylin. Positive cells within 100 randomly selected glomeruli per animal were enumerated by light microscopy in a blinded fashion (25). Intraglomerular infiltration was quantitated by calculating the average number of CAE+ cells per glomerulus.

Immunofluorescence

Fresh kidney tissue from Expt. A and B was mounted in OCT compound (Miles Diagnostics, Elkhart, IN) and frozen in liquid isopentane cooled on dry ice, and 4-µm cryostat sections were prepared (Leica, Nussloch, Germany). Direct immunofluorescence studies were performed by fixing frozen sections in acetone followed by blocking with 10% normal goat serum and addition of FITC-conjugated sheep Ab directed against rabbit IgG (Zymed, South San Francisco, CA), FITC-conjugated rabbit Ab directed against human fibrinogen (Dako, Copenhagen, Denmark), or FITC-conjugated rat anti-mouse CD11b (Harlan/Serotec, Indianapolis, IN) at a 1/100 dilution. After washing with PBS and 1% Tween-20, sections were studied under a fluorescence microscope by a pathologist (F.K.B.) in a blinded fashion. The average number of intracapillary fibrinogen deposits and intraglomerular CD11b+ cells was determined by examining 100 glomeruli/animal (20).

RNA isolation and RT-PCR

Kidney tissue obtained in Expt. A was snap-frozen in liquid nitrogen and stored at -80°C. Total kidney RNA was extracted in guanidinium salt solution and purified by the cesium chloride method (27). RNA samples with OD260/280 <1.6 were rejected. Five micrograms of total RNA was reverse transcribed using oligo(dT) primers and Superscript reverse transcriptase according to the manufacturer’s instructions (Life Technologies, New Haven, CT). Ten percent of cDNA was then subjected to 38 cycles of PCR amplification (total volume of reaction, 100 µl) in a Perkin-Elmer Thermocycler 480 (Perkin-Elmer, Foster City, CA) using mouse IL-4-specific primers. RT-PCR controls included "no RNA" (blank) and "no reverse transcriptase" reactions. Fifteen microliters of each RT-PCR reaction was electrophoresed on 2% SeaKem LE agarose (American Bioanalytical, Natick, MA) gels and stained with ethidium bromide. RT-PCR of a housekeeping gene (HPRT) was performed to verify equal RNA and cDNA loading in the RT and PCR reactions, respectively.

Northern blotting

Total kidney RNA (20 µg/sample) was electrophoresed on 1% formaldehyde-agarose gels, transferred to Hybond-N+ membranes (Amersham Life Sciences, Arlington Heights, IL), and covalently linked by UV irradiation (Stratagene, La Jolla, CA). Radiolabeled probe was prepared using 50 ng of full-length murine intercellular adhesion molecule-1 (ICAM-1) cDNA (American Type Culture Collection, Rockville, MD) in a standard oligolabeling protocol (Pharmacia Biotech, Piscataway, NJ) using [32P]dCTP (Amersham). Approximately 1 to 2 x 106 cpm of labeled probe (sp. act. = ~1 x 109 dpm/µg DNA) were used per milliliter of hybridization solution. Hybridizations were performed at 42°C for 16 h, following which membranes were washed at a final stringency of 0.1x SSC/0.1% SDS at 55°C. Autoradiography was performed using Hyperfilm-MP (Amersham) and an intensifying screen at -70°C for 12 h. Membranes were then stripped and reprobed with GAPDH radiolabeled cDNA according to the above procedure. Densitometric analysis of Northern blots was performed using a Molecular Dynamics densitometer (Sunnyvale, CA).

Statistical analyses

Analysis of variance (Fisher’s protected least significant difference and/or Scheffe’s test) or unpaired t test was used to compare differences between groups. The confidence interval was set at 95%.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Intrarenal IL-4 mRNA expression in murine anti-GBM nephritis

Complete inactivation of IL-4 gene function in IL-4-/- mice was verified by measuring IL-4 protein concentration in supernatants of Con A-stimulated splenocytes. While IL-4+/+ splenocytes produced up to 32 pg/ml of the cytokine over 48 h, IL-4 was not detected (<5 pg/ml) in IL-4-/- supernatants. We then studied intrarenal IL-4 mRNA expression before and after NTS injection. As shown in Figure 1Go, IL-4 mRNA measured by RT-PCR was present in normal IL-4+/+ renal tissue and increased significantly 2 h following induction of nephritis. This is consistent with our previous observation that intrarenal IL-4 mRNA and IL-4 protein are up-regulated in rat anti-GBM nephritis (25). On the other hand, IL-4-/- mice failed to express IL-4 mRNA in their kidneys in either the basal state or after induction of nephritis (Fig. 1Go).



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  		FIGURE 1. RT-PCR analysis of intrarenal IL-4 mRNA expression before (pre-NTS) and 2 h after nephrotoxic serum administration (postNTS). Total RNA was prepared from IL-4+/+ (+/+) and IL-4-/- (-/-) kidneys, reverse transcribed, and subjected to 38 cycles of PCR. Fifteen percent of each PCR reaction was electrophoresed on 2% agarose gels and stained with ethidium bromide. Results were similar in three separate animal experiments. L, DNA ladder ({phi}X174RF DNA/HaeIII fragments); C1, control PCR reaction without reverse transcriptase; C2, control PCR reaction without cDNA.

 
IL-4 is an endogenous suppressor of neutrophil infiltration in murine anti-GBM nephritis

The acute inflammatory response in anti-GBM nephritis is characterized by intraglomerular neutrophil accumulation that peaks 2 h after disease induction (20). To determine whether IL-4 is an endogenous regulator of this response, intraglomerular neutrophil (CAE+ cells) counts in IL-4+/+ and IL-4-/- mice were compared 2 h after NTS injection. As shown in Figure 2Go, NTS induced significantly greater neutrophil infiltration in IL-4-/- (mean ± SE = 1.7 ± 0.4 CAE+ cell/glomerulus) than in IL-4+/+ glomeruli (0.5 ± 0.2 CAE+ cell/glomerulus; p < 0.05). Administration of recombinant murine IL-4 at the time of NTS injection reduced intraglomerular neutrophil accumulation in IL-4-/- kidneys to levels comparable to those in the IL-4+/+ group (0.5 ± 0.1 and 0.5 ± 0.2 CAE+ cell/glomerulus, respectively; Fig. 2Go). Neutrophil counts before NTS injection (pre-NTS) were similar in IL-4+/+ and IL-4-/- glomeruli (0.05 ± 0.02 and 0.08 ± 0.02 CAE+ cell/glomerulus, respectively; Fig. 2Go). Control IL-4+/+ and IL-4-/- mice injected with NRS did not display significant neutrophil accumulation in their glomeruli.



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  		FIGURE 2. Average intraglomerular neutrophil counts before (pre-NTS) and 2 h after nephrotoxic serum administration (post-NTS) in IL-4+/+ ({blacksquare}), IL-4-/- ({square}), and IL-4-/- mice treated with recombinant murine IL-4 at the time of NTS injection (). Neutrophils were identified by CAE histochemical staining, and the average number of neutrophils per glomerulus (mean ± SD) was determined by inspecting 100 glomeruli in each kidney sample. * indicates significantly greater than value in either IL-4+/+ or IL-4-treated IL-4-/- mice (p < 0.05).

 
IL-4 is an endogenous suppressor of ICAM-1 up-regulation in murine anti-GBM nephritis

To investigate the mechanism by which IL-4 inhibits neutrophil migration, we studied intrarenal ICAM-1 mRNA expression before and 2 h after NTS administration. ICAM-1 plays an important role in neutrophil adherence to the vascular endothelium (28). As shown in three separate animal experiments (Fig. 3GoA), ICAM-1 mRNA up-regulation following induction of nephritis was significantly greater in IL-4-/- than in IL-4+/+ kidneys. Quantitative densitometric analysis of ICAM-1 mRNA revealed a 3.1 ± 0.3-fold increase over baseline in IL-4+/+ kidneys compared with a 5.7 ± 0.9-fold increase in the IL-4-/- group (mean ± SD; p < 0.05; Fig. 3GoB). IL-4 administration at the time of NTS injection reduced intrarenal up-regulation of ICAM-1 mRNA in IL-4-/- mice to levels observed in IL-4+/+ mice (3.2 ± 0.4- vs 3.1 ± 0.3-fold increase, respectively; Fig. 3Go, A and B). Baseline (pre-NTS) expression of ICAM-1 did not differ significantly between the IL-4+/+ and IL-4-/- groups (Fig. 3GoA).



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  		FIGURE 3. Northern blot analysis of intrarenal ICAM-1 mRNA expression before (pre-NTS) and 2 h after nephrotoxic serum administration (postNTS). A, Total RNA was prepared from kidneys of IL-4+/+ (+/+), IL-4-/- (-/-), or IL-4-/- mice treated with recombinant murine IL-4 at the time of NTS injection (-/-*). One percent formaldehyde-agarose RNA gels from three separate animal experiments were probed with 32P-labeled murine ICAM-1 cDNA. One representative gel reprobed with 32P-labeled murine GAPDH cDNA is also shown. B, Densitometric analysis of intrarenal ICAM-1 mRNA expression; IL-4+/+ ({blacksquare}), IL-4-/- ({square}), and IL-4-/- mice treated with recombinant murine IL-4 at the time of NTS injection () are shown. The fold increase over baseline (mean ± 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. {ddagger}, Significantly greater than value in either IL-4+/+ or IL-4-treated IL-4-/- mice (p < 0.05).

 
Increased glomerular pathology in IL-4-/- mice after induction of murine anti-GBM nephritis

Functional and morphologic renal abnormalities in murine anti-GBM nephritis develop within several hours to a few days after neutrophil influx into glomerular tissue (20, 21). These abnormalities are neutrophil dependent and include proteinuria, increased glomerular cellularity, and fibrinogen deposits within glomerular capillaries and mesangium (22, 23, 24). We, therefore, investigated whether the glomerular lesions of anti-GBM nephritis are exaggerated in the absence of IL-4. There were no deaths following NTS injection. Average body weights in the IL-4+/+ and IL-4-/- groups were similar at the end of the experiment (27 ± 3 and 28 ± 2 g, respectively). Intense linear staining for rabbit IgG could be demonstrated along the GBM after NTS administration and did not differ between IL-4+/+ and IL-4-/- glomeruli (data not shown). Circulating mouse anti-rabbit IgG or intrarenal mouse IgG deposits were not detected. Increased protein excretion was not observed in either group at 2 h. By the fourth day of nephritis, however, IL-4-/- mice displayed worse glomerular injury than IL-4+/+ mice, as measured by the following parameters.

Proteinuria (Up/UCr). Urinary protein excretion in the IL-4-/- group on days 1 and 4 after NTS injection was higher than that in day-matched IL-4+/+ mice (Fig. 4Go). The difference in Up/UCr ratio on day 4 between the IL-4-/- group (mean ± SE = 71 ± 6; n = 10) and the IL-4+/+ group (41 ± 7; n = 10) reached statistical significance (p < 0.05; Fig. 4Go). Administration of recombinant murine IL-4 (1000 U i.p., twice per day) to IL-4-/- mice starting at the time of NTS injection reduced the day 4 Up/UCr ratio to 50 ± 10 (n = 4), which was not significantly different from that in the IL-4+/+ group. Urinary protein excretion did not increase over baseline in control mice injected with NRS.



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  		FIGURE 4. Average proteinuria (mean ± SE), represented by the Up/UCr, in IL-4+/+ (•; n = 10) and IL-4-/- ({circ}; n = 10) mice on days 0, 1, and 4 after injection of nephrotoxic serum (postNTS). * indicates significantly greater than value in day-matched IL-4+/+ mice (p < 0.05).

 
Histopathologic changes. Four days following NTS injection, the majority of IL-4-/- mice (8 of 10) developed diffuse renal pathology characterized by glomerular hypercellularity and narrowing or obliteration of capillary lumina due to intracapillary PAS+ deposits (Fig. 5GoB). Glomerular enlargement, glomerular neutrophil infiltrate, intramesangial PAS+ deposits, and intratubular proteinacious casts were also observed on light microscopic examination. On the other hand, only 3 of 10 IL-4+/+ mice had significant glomerular changes that resembled those observed in the IL-4-/- group. The remaining IL-4+/+ mice had normal renal histology except for focal intraglomerular PAS+ deposits (Fig. 5GoA). Average glomerular cellularity and neutrophil infiltrate in IL-4+/+ kidneys 4 days after NTS administration were significantly higher than those in IL-4-/- kidneys (Table IGo). Baseline (pre-NTS) glomerular cellularity was not different between the two groups (Table IGo). We also observed that IL-4-/- mice developed significantly more intracapillary fibrinogen deposits per glomerulus than IL-4+/+ mice (Table IGo). Occlusion of intraglomerular capillaries with fibrinogen is a central feature of murine anti-GBM nephritis (20, 21).



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  		FIGURE 5. Glomerular histopathology 4 days after induction of nephritis in IL-4+/+ (A) and IL-4-/- (B) mice. PAS-stained tissue sections are shown (x400 magnification). Arrows point to intracapillary PAS+ deposits, which are prominent in IL-4-/- glomeruli. These deposits were identified as fibrinogen by immunofluorescence.

 

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  		Table I. Glomerular histology in IL-4+/+ and IL-4-/-

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Neutrophil accumulation in glomerular tissue is a hallmark of acute, Ab-induced glomerulonephritis (16). Their transmigration into glomeruli is facilitated by ICAMs expressed on endothelial cells (29). Neutrophil depletion (30, 31) or inhibiting neutrophil migration by blocking their adherence to the endothelium (32, 33) ameliorates experimental Ab-induced glomerulonephritis. In this study we provided direct evidence that IL-4 is an endogenous suppressor of intrarenal ICAM-1 mRNA up-regulation, neutrophil influx, and subsequent glomerular pathology in murine anti-GBM nephritis. Previous reports have shown that IL-4 protects against glomerulonephritis in lupus-prone mice by down-regulating nephrotoxic subclasses of IgG autoantibodies (18, 19). Our results demonstrate that IL-4 also exerts anti-inflammatory actions following binding of nephrotoxic Abs to kidney tissue.

The observation that endogenous IL-4 inhibits neutrophil influx is consistent with known pharmacologic effects of this cytokine. Mulligan et al. reported that intratracheal administration of rIL-4 suppresses neutrophil infiltration in a murine model of immune complex-mediated lung injury (14). Similarly, i.v. IL-4 inhibited neutrophil accumulation in a murine air pouch after local application of IL-1ß (34). We also observed in this study that IL-4 is an endogenous inhibitor of ICAM-1 mRNA up-regulation following induction of murine anti-GBM nephritis. It is likely that intrarenal ICAM-1 mRNA correlates with ICAM-1 protein expression in this model because NTS administration has been shown to increase ICAM-1 on glomerular endothelial cells (33, 35). Mulligan et al. demonstrated that exogenous IL-4 suppresses immune complex-induced ICAM-1 up-regulation in pulmonary vessels (14). Similarly, in vitro experiments have shown that IL-4 inhibits IL-1ß-induced ICAM-1 up-regulation on endothelial cells (36, 37). Taken together, these observations suggest that modulation of ICAM-1 expression is an important mechanism by which IL-4 inhibits neutrophil influx. It is possible, however, that IL-4 suppresses neutrophil migration by additional mechanisms. For example, IL-4 inhibits IL-8 production by monocytes and neutrophils (11, 38). In vivo neutralization of IL-8 has been shown to prevent neutrophil influx in Ab-mediated glomerulonephritis (39). IL-4 also inhibits leukotriene B4 production by monocytes (40). Leukotriene B4 is a potent neutrophil chemoattractant (41) and may play a role in recruiting neutrophils to the kidney in anti-GBM nephritis (42).

We also observed in this study that IL-4-/- mice develop worse glomerular pathology than the IL-4+/+ group. Four days after NTS administration, IL-4-/- mice had significantly greater proteinuria, glomerular cellularity, and intracapillary fibrinogen deposits. Because neutrophils play a central role in murine anti-GBM nephritis (22, 23, 24), increased pathology in IL-4-/- mice could have resulted from greater neutrophil influx into their glomeruli. We did not detect significant monocyte (CD11b+ cell) accumulation in either IL-4+/+ or IL-4-/- kidneys (less than one cell per glomerulus 4 days after NTS injection). Others have shown that T lymphocytes and monocytes do not contribute significantly to glomerular pathology in this model (43, 44, 45). The difference in proteinuria between IL-4-/- and IL-4+/+ mice on the first day of nephritis was less marked than the difference in neutrophil accumulation between the two groups. This discrepancy may be explained by neutrophil-independent mechanisms of proteinuria such as nephrotoxic Abs, which directly alter the glomerular filtration barrier (46, 47). We suspect that glomerular hypercellularity in IL-4-/- mice resulted in part from increased mesangial cell number because mesangial cell proliferation has been observed in experimental Ab-induced glomerulonephritis (48), and IL-4 has been shown to suppress proliferation of mesangial cells in culture (49).

Our results do not exclude that IL-4 can modulate glomerulonephritis by down-regulating the inflammatory functions of endothelial cells and resident macrophages. For example, IL-4 suppresses procoagulant expression by endothelial cells (12) and inhibits nitric oxide production by macrophages (50). It remains to be determined whether procoagulant activity and nitric oxide release are increased in IL-4-/- glomeruli and whether they contribute to intracapillary thrombosis in murine anti-GBM nephritis. IL-4 also stimulates IL-1RA while inhibiting IL-1 and TNF-{alpha} production by monocytes (5, 51). In addition, it induces IL-1 decoy receptor synthesis by neutrophils (52). Although a balance between IL-1 and its antagonists may determine the outcome of some forms of experimental glomerulonephritis (53, 54), their role in murine anti-GBM nephritis is not known.

In conclusion, we demonstrated that endogenously produced IL-4 down-regulates ICAM-1 expression, neutrophil influx, and subsequent pathology in murine Ab-induced glomerulonephritis. In contrast to IL-4’s role in promoting IgE-mediated immediate-type hypersensitivity, these results suggest that IL-4 is an inhibitor of acute inflammation induced by IgG Abs.


   Acknowledgments
 
The authors thank Dr. Manfred Kopf (Basel Institute of Immunology, Basel, Switzerland) and Dr. Osami Kanagawa (Washington University, St. Louis, MO) for providing IL-4 gene knockout mice, and Dr. Satwant Narula (Schoering-Plough Research Institute, Kenilworth, NJ) for providing recombinant murine IL-4.


   Footnotes
 
1 This work was supported by a Veterans Affairs Merit Review grant (to F.G.L.). Back

2 Address correspondence and reprint requests to Dr. Fadi G. Lakkis, Emory University and Veterans Affairs Medical Center, Research 151N, 1670 Clairmont Rd., Atlanta, GA 30033. E-mail: Back

3 Abbreviations used in this paper: GBM, glomerular basement membrane; NTS, nephrotoxic serum; NRS, nonimmune rabbit serum; Up/UCr, ratio of urinary protein to creatinine concentration; PAS, periodic acid-Schiff; CAE, chloroacetate esterase; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ICAM-1, intercellular adhesion molecule-1. Back

Received for publication June 11, 1997. Accepted for publication October 3, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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