J Am Soc Nephrol 12: 464–471, 2001 -12 from Intrinsic Cells Is an Effector of Renal Injury in Crescentic Glomerulonephritis

JENNIFER R. TIMOSHANKO, A. RICHARD KITCHING, STEPHEN R. HOLDSWORTH, and PETER G. TIPPING From the Center for Inflammatory Diseases, Monash University, Department of Medicine, Monash Medical Center, Clayton, Victoria, Australia.

Abstract. Interleukin-12 (IL-12) directs the cognate nephrito- paired renal function (elevated proteinuria and serum creati- genic T helper type 1 responses that initiate renal injury in nine) 10 d after initiation of GN. IL-12 Ϫ/Ϫ mice showed murine crescentic glomerulonephritis (GN). The recent dem- significant protection from GN. Chimeric IL-12 mice showed onstration of IL-12 production by intrinsic renal cells, includ- significant attenuation of crescent formation, glomerular T-cell ing mesangial and proximal tubular cells, raises the possibility and accumulation, and renal impairment, com- that IL-12 from nonimmune cells may contribute to inflamma- pared with WT and sham chimeric mice, but were not pro- tory renal injury. To address this possibility, the development tected to the same extent as IL-12 Ϫ/Ϫ mice. IL-12 chimeric of sheep anti-mouse glomerular basement membrane globulin– mice showed no attenuation of their systemic cognate immune induced crescentic GN was studied in C57BL/6 wild-type response to the nephritogenic antigen (sheep globulin), indi- (WT), IL-12–deficient (IL-12 Ϫ/Ϫ), and IL-12 “chimeric” cated by antigen-specific circulating antibody and cutaneous mice. IL-12 chimeric mice were produced by transplantation of delayed-type hypersensitivity. These studies indicate that WT bone marrow into IL-12 Ϫ/Ϫ mice to restore IL-12 pro- IL-12 produced by non–bone marrow derived intrinsic renal duction by immune cells, while leaving them deficient in renal cells contributes to immune renal injury. They provide the first IL-12 production. WT and “sham” chimeric mice (normal bone in vivo demonstration of a proinflammatory role for an intrinsic marrow transplanted into WT mice) developed crescentic GN renal cell–derived in renal inflammation. with glomerular T-cell and macrophage recruitment and im-

Glomerular crescent formation is a feature of the most severe GN. Augmentation of Th1 responses by administration of forms of human glomerulonephritis (GN). The association of T IL-12 (8) or genetic absence of IL-4 (10) or IL-10 (11) exac- cells and , proliferation of glomerular epithelial erbates crescentic GN. cells, and fibrin deposition in human (1,2) and experimental IL-12 is a heterodimeric cytokine composed of two co- crescentic GN (3,4) suggests that this form of glomerular valently linked subunits, p35 and p40, encoded by separate inflammation results from a delayed-type hypersensitivity genes (12). Macrophages and produce IL-12 as an (DTH)-like cell-mediated, cognate immune response. Glomer- early response to antigenic stimuli (13). Antigen-presenting ϩ ular injury has been demonstrated to be dependent on CD4 T cells produce IL-12 after engagement with activated T cells ϩ cells but not on CD8 T cells or autologous Ig in murine and co-stimulation via CD40 ligand (14). IL-12 primes CD4ϩ crescentic GN (4,5). Studies in planted antigen and autoim- T cells for high IFN-␥ production (14,15) and polarizes un- mune models of crescentic GN indicate that Th1 subset re- committed T cells toward a Th1 profile. The role of IL-12 in sponses are pivotal in directing crescentic glomerular injury. T immune responses in vivo has been referred to as a “functional helper type 1 (Th1)-prone strains of mice are more susceptible bridge” between the early noncognate innate resistance and to crescentic injury than are Th2-prone strains (4,6). Attenua- subsequent antigen-specific adaptive immunity (13,16,17). tion of the primary Th1 immune response by immunological IL-12 has been demonstrated in crescentic glomeruli of mice ␥ inhibition or genetic deletion of Th1 - that are susceptible to autoimmune anti–glomerular basement ␥ (IFN- ) (4,7) and interleukin-12 (IL-12) (8) or by administra- membrane (anti-GBM)-induced GN but was not detectable in tion of Th2 cytokines IL-4 and IL-10 (9) attenuates crescentic strains that are resistant to the development of crescentic glo- merular lesions (6). In the autoimmune GN associated with the “lupus like” syndrome of MRL/Faslpr mice, intrarenal IL-12 Received December 14, 1999. Accepted July 19, 2000. expression by infiltrating mononuclear cells and tubular epi- Correspondence to Dr. Peter G. Tipping, Monash University, Department of thelial cells was demonstrated in association with increased Medicine, Monash Medical Center, 246 Clayton Road, Clayton, 3168, Victo- IFN-␥ mRNA (18). In vitro, renal tubular epithelial cells pro- ria, Australia. Phone: 61 3 9594 5547; Fax: 61 3 9594 4279; E-mail: [email protected] duce IL-12 mRNA and low levels of IL-12 protein (18), and 1046-6673/1203-0464 mesangial cells produce IL-12 mRNA and protein after lipo- Journal of the American Society of Nephrology polysaccharide or -␣ stimulation (19). Copyright © 2001 by the American Society of Nephrology Mesangial cells also express the IL-12 ␤1 chain and J Am Soc Nephrol 12: 464–471, 2001 Intrinsic IL-12 Augments Renal Injury 465

respond directly to IL-12 stimulation by production of platelet- previously (22). Male WT, IL-12–deficient (IL-12 Ϫ/Ϫ), bone mar- activating factor and reactive oxygen species (19). Therefore, row transplanted WT (sham chimeric), and bone marrow transplanted in addition to the critical role for IL-12 in directing nephrito- IL-12 Ϫ/Ϫ (IL-12 chimeric) mice, all 13 to 14 wk of age, were ␮ genic Th1 responses, IL-12 produced by intrinsic renal cells sensitized by subcutaneous injection of 100 g of sheep globulin in ␮ has the capacity to act as an effector cytokine in crescentic GN. 100 l of CFA. Ten d later, GN was initiated by intravenous admin- istration of 4.4 mg sheep anti-mouse GBM globulin. The development The current studies evaluated the capacity of IL-12 produced of GN was assessed 10 d after administration of anti-GBM globulin. by intrinsic renal cells to act as a local effector molecule in Th1-dependent cell-mediated renal inflammation by studying the development of crescentic GN in “IL-12 chimeric” mice Histologic Assessment of Glomerular Injury with absent intrinsic renal cell IL-12 production but normal Glomerular Crescent Formation. Kidney tissue was fixed in IL-12 production from bone marrow-derived immune/inflam- Bouin’s fixative and embedded in paraffin, and 3-␮m sections were matory cells. stained with periodic acid-Schiff (PAS) reagent. Glomeruli were considered to exhibit crescent formation when two or more layers of cells were observed in Bowman’s space. A minimum of 50 glomeruli Materials and Methods were assessed to determine the crescent score for each animal. Mice Glomerular T-Cell and Macrophage Accumulation. Kidney Breeding pairs of mice with targeted disruption of the IL-12 p40 tissue was fixed in periodate/lysine/paraformaldehyde, sectioned, and gene (IL-12 Ϫ/Ϫ) (20), which have been backcrossed for nine gen- stained in an identical manner as that described for spleen to demon- erations onto a C57BL/6 background, were obtained from Jackson strate CD4ϩ T cells and macrophages. A minimum of 20 equatorial Laboratories (Bar Harbor, ME). C57BL/6 mice were used as wild- sectioned glomeruli were assessed per animal, and results were ex- type (WT) controls, and all mice were housed and bred under specific pressed as cells per glomerular cross section (c/gcs). pathogen free (SPF) conditions at Monash University (Clayton, Vic- Tubulointerstitial Infiltration. The number of interstitial cells toria, Australia). All animal experimentation described in this article was counted by means of a 10-mm2 graticule fitted in the eyepiece of was conducted in accordance with the Monash Medical Center Ani- the microscope. Five randomly selected cortical areas, which ex- mal Ethics Committee guidelines. cluded glomeruli, were counted for each animal. Each high-power field represented an area of 1 mm2, and counts were performed in a 2 Bone Marrow Transplantation blinded protocol. Data are expressed as cells/mm and represent the Ϯ Five- to 6-wk-old male recipient IL-12 Ϫ/Ϫ or C57BL/6 mice mean SEM for animals in each group. received 1100 rads of total body irradiation. Bone marrow cells were harvested aseptically from the femora and tibiae of SPF WT donor Functional Assessment of Glomerular Injury ϫ 6 mice, and red blood cells were lysed. Recipient mice received 5 10 Mice were housed individually in cages to collect urine before nucleated cells intravenously within6hofirradiation. Mice were administration of anti-GBM globulin and over the final 24 h of the maintained under SPF conditions for 8 wk to allow bone marrow experiment. Urinary protein concentrations were determined by a reconstitution. modified Bradford method (23). Serum creatinine concentrations were measured by the alkaline picric acid method using an autoanalyzer Assessment of Bone Marrow Engraftment and (Cobas Bio, Roche Diagnostic, Basel, Switzerland). Lymphocyte Subset Reconstitution Circulating leukocyte numbers and lymphocyte subsets were as- Histologic Demonstration of IL-12 Expression sessed 8 wk after bone marrow transplantation. Cryostat cut snap-frozen kidney tissue sections (6 ␮m) were Circulating Leukocyte Numbers. Blood was collected into stained for IL-12 and macrophages by direct immunofluorescence 3.3% sodium citrate, and circulating leukocyte numbers were deter- using mAb conjugated with Alexa Fluor dyes. Rat anti-mouse IL-12 mined by counting in a hemocytometer after lysis of red blood cells. mAb (anti–IL-12 p40, clone 15.6, a gift of Dr. G. Trinchieri, Wistar Circulating Lymphocyte Subsets. Citrated blood was incubated Institute, Philadelphia, PA) was conjugated with Alexa Fluor 594 dye with FITC-conjugated anti-mouse CD4, anti-mouse CD8, and anti- (Molecular Probes, Eugene, OR, absorption 590 nm, and fluorescence B220 monoclonal antibodies (mAb; PharMingen, San Diego, CA), emission 617 nm equivalent to the spectra for Texas Red). Rat and lymphocyte subsets were quantified by flow cytometry, as de- anti-mouse macrophage mAb M 1/70 was conjugated with Alexa scribed previously (21). Fluora dye 488 (Molecular Probes, absorption 494 nm, and fluores- Lymphocyte Subset and Macrophage Repopulation in the cence emission 519 nm equivalent to the spectra for FITC). Staining Spleen. Splenic tissue was fixed in periodate/lysine/paraformalde- of tissue from IL-12–deficient mice provided a negative control. hyde for 4 h, washed in 7% sucrose, frozen in liquid nitrogen, and Sections were incubated concurrently with both antibodies at a final stored at Ϫ70°C. Tissue sections (6 ␮m) were stained to demonstrate dilution of 1:50 for 60 min at room temperature and were examined by T cells and macrophages using a three-layer immunoperoxidase tech- confocal microscopy. Confocal images were collected using a confo- nique as described previously (3). The primary antibodies were GK1.5 cal inverted Nikon Diaphot 300 microscope (Bio-Rad, Hercules, CA) (anti-mouse CD4 mAb, American Type Tissue Culture Collection equipped with an air-cooled 25-mWat argon/krypton laser with lines [ATCC], Manassas, VA) and M 1/70 (anti-mouse Mac-1 mAb, at 488, 586, and 647 nm and triple dichroic and 560 dichroic long pass ATCC). filter sets. Digital images were produced from an average of 8-line scans of approximately 1 s duration with the oil immersion lens ϫ40 Induction of GN and were collected by using a Pentium 90 computer scan control and Anti-GBM globulin was prepared from serum of a sheep immu- the image acquisition and image analysis software packages COS- nized against a particulate fraction of mouse GBM as described MOS (Bio-Rad). 466 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 464–471, 2001

Assessment of the Systemic Immune Response to Sheep are expressed as the mean Ϯ SEM. The statistical significance was Globulin determined by one-way ANOVA, followed by Fisher’s protected least Cutaneous DTH. Sensitized mice that developed GN were chal- significant difference post hoc analysis. lenged 24 h before the end of the experiment by intradermal injection of 500 ␮g of sheep globulin in 50 ␮l of phosphate-buffered saline into Results the plantar surface of a hind footpad. A similar dose of an irrelevant IL-12 Deficiency Protects against Development of antigen (Ag; horse globulin) was injected into the opposite footpad as Crescentic GN and Cutaneous DTH a control. Footpad swelling was quantified 24 h later using a micro- Normal renal histology was unaffected by bone marrow meter. Ag-specific DTH was taken as the difference in skin swelling between sheep globulin– and horse globulin–injected footpads and transplantation (Figure 1, A through C). Sensitized WT mice expressed ⌬ footpad thickness (mm). developed proliferative GN with crescent formation and inter- Measurement of Circulating Mouse Anti-Sheep Antibody. stitial inflammatory infiltration 10 d after administration of ϩ Mouse anti-sheep globulin antibody (Ab) titers were measured by anti-GBM globulin (Figure 1D). CD4 cells (Figure 2A) and enzyme-linked immunosorbent assay on serum collected at the end of macrophages (Figure 2B) were observed in glomeruli. IL-12 each experiment as described previously (7). Serial dilutions of serum expression was not detectable in glomeruli of normal WT mice were assayed using sheep globulin (10 ␮g/ml) as the capture Ag and but was expressed in glomeruli and tubules after induction of horseradish peroxidase–conjugated sheep anti-mouse Ig Ab (Amer- GN (Figure 2, C and D). In WT mice, glomerular IL-12 sham, Little Chalfont, UK) as the detecting Ab. expression was observed in association with intraglomerular macrophages but also in areas where macrophages were not Statistical Analyses present. IL-12 expression did not co-localize with periglomeru- Assessment of bone marrow engraftment by analysis of circulating lar macrophages (Figure 2D) or with interstitial macrophages lymphocyte subsets was performed on two separate occasions in a (Figure 2C). IL-12–deficient mice showed significant protec- total of 11 IL-12 chimeric and 8 sham chimeric mice. Age-matched tion against development of crescentic GN compared with WT WT mice (n ϭ 6) served as controls. The development of GN was studied in groups of WT, IL-12 chimeric, and sham chimeric mice on mice (Figure 1F), and IL-12 was undetectable in their glomer- two separate occasions and on a single group of IL-12 Ϫ/Ϫ mice. All uli (Figure 2F). The incidence of crescent formation (IL-12 mice were males between 13 and 14 wk of age. The total numbers of Ϫ/Ϫ,7Ϯ 0.6% of glomeruli; WT, 32 Ϯ 3%; P Ͻ 0.0001) and ϩ mice with GN in each group were as follows: WT, n ϭ 6; IL-12 glomerular infiltration of CD4 T cells (IL-12 Ϫ/Ϫ, 0.5 Ϯ 0.1 chimera, n ϭ 8; sham chimera n ϭ 5; and IL-12 Ϫ/Ϫ, n ϭ 4. Results c/gcs; WT, 1.2 Ϯ 0.1; P Ͻ 0.0001) and macrophages (IL-12

Figure 1. Photomicrographs demonstrating the histologic appearances of kidneys from wild type (WT; A), interleukin-12 (IL-12) chimeric (B), and IL-12–deficient (C) mice before induction of glomerulonephritis (GN). After induction of GN, WT mice developed proliferative GN with prominent crescent formation (D). Proliferative GN was less severe and crescent formation was less frequent in IL-12 chimeric mice compared with WT mice (E), and only mild glomerular hypercellularity was apparent in IL-12–deficient mice (F). Magnifications: ϫ200 in A through C (periodic acid-Schiff [PAS] stain); ϫ400 in D through F (PAS stain). J Am Soc Nephrol 12: 464–471, 2001 Intrinsic IL-12 Augments Renal Injury 467

Figure 2. Photomicrographs demonstrating CD4ϩ T cells (A) and macrophages (B) in glomeruli of WT mice with GN. Confocal images demonstrating IL-12 expression (red) on tubules (C) and in glomeruli of WT mice with GN (D). Co-localization of IL-12 (yellow) on the surface of intraglomerular macrophages (green) can be seen (arrows). Periglomerular macrophages did not express IL-12. In chimeric mice (E), IL-12 expression was restricted to areas adjacent to intraglomerular macrophages (arrows). IL-12 expression could not be detected in IL-12–deficient mice after induction of GN (F). Magnification, ϫ400 (immunoperoxidase, hematoxylin counterstain in A through C; confocal immunofluo- rescent images under oil immersion in D through F).

Ϫ/Ϫ, 1.1 Ϯ 0.1 c/gcs; WT, 3.3 Ϯ 0.4 c/gcs; P Ͻ 0.0001) were T-cell subsets, and B cells in IL-12 and “sham” chimeras were significantly reduced (Figure 3). Reduction in the histologic equivalent to those in WT mice (Table 1). Their splenic archi- appearances of renal injury and the glomerular mononuclear tecture with regard to distribution of CD4ϩ T cells and mac- and interstitial cell infiltrate (IL-12 Ϫ/Ϫ, 64.5 Ϯ 3.6 cells/ rophages was normal (data not shown). After cutaneous sen- mm2; WT, 141.3 Ϯ 4.2 cells/mm2; P Ͻ 0.001) was associated sitization and intravenous administration of sheep globulin, with significant protection of renal function. This was indi- IL-12 and sham chimeric mice showed identical systemic cated by reductions in proteinuria (IL-12 Ϫ/Ϫ, 1.2 Ϯ 0.1 immune responses to the nephritogenic antigen as WT mice. mg/24 h; WT, 3.9 Ϯ 0.8 mg/24 h; P Ͻ 0.005) and serum The serum levels of mouse anti-sheep globulin Ab were iden- creatinine (IL-12 Ϫ/Ϫ,20Ϯ 2 ␮mol/L; WT, 35 Ϯ 3 ␮mol/L; tical (Figure 5, and cutaneous DTH after antigen challenge in P Ͻ 0.005; Figure 4). IL-12 chimeras (Ag-specific skin swelling, 0.74 Ϯ 0.05 mm) IL-12–deficient mice showed attenuation of DTH to the was equivalent to that in WT (0.81 Ϯ 0.07 mm) and sham nephritogenic antigen as indicated by significantly reduced chimeric mice (0.66 Ϯ 0.05 mm). This indicated functional cutaneous swelling after an intradermal challenge with sheep restoration of immune competence in engrafted mice and sug- globulin (IL-12 Ϫ/Ϫ, 0.28 Ϯ 0.03 mm; WT, 0.81 Ϯ 0.07 mm; gests that IL-12 production by non–bone marrow–derived P Ͻ 0.001). The serum titers of autologous anti-sheep globulin cells is not required for the full expression of these humoral Ab were similar in WT- and IL-12–deficient mice (Figure 5), and cell mediated systemic responses. indicating that the humoral immune response to the nephrito- genic antigen was unaffected by IL-12 deficiency. IL-12 Production by Intrinsic Renal Cells Is Required for Full Expression of Crescentic GN “Chimeric” IL-12 Mice Have Normal Lymphocyte Sham chimeric mice developed crescentic GN, with similar Subsets and Normal Systemic Immune Responses to histologic features to WT mice with GN, indicating that bone Sheep Globulin marrow transplantation per se did not affect the development Bone marrow engraftment, studied 8 wk after transplanta- of this disease. The incidences of crescentic glomeruli (WT, 31 tion, demonstrated that circulating white blood cell numbers, Ϯ 3%; sham chimeras, 33 Ϯ 1%) and glomerular infiltration of 468 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 464–471, 2001

Figure 3. The incidence of crescent formation (% glomeruli affected) and glomerular infiltration of CD4ϩ T cells and macrophages (cells/ per glomerular cross-section [c/gcs]) in normal (WT), sham chimeric, IL-12 chimeric, and IL-12–deficient (IL-12 Ϫ/Ϫ) mice developing crescentic glomerulonephritis. Figure 4. Proteinuria (mg/24 h), serum creatinine (␮mol/L), and interstitial cell counts (cells/mm2) in WT, sham chimeric, IL-12 chimeric, and IL-12–deficient (IL-12 Ϫ/Ϫ) mice developing GN. The CD4ϩ T cells (WT, 1.2 Ϯ 0.1 c/gcs; sham chimeras, 1.1 Ϯ 0.05 dotted line represents the mean proteinuria, creatinine, and interstitial c/cgs) and macrophages (WT, 3.3 Ϯ 0.4 c/gcs; sham chimeras, infiltrate in normal mice. 3.2 Ϯ 0.1 c/gcs) were equivalent (Figure 3). Similarly, there was no difference in functional renal injury between the two groups, indicated by proteinuria (WT, 3.9 Ϯ 0.8 mg/24 h; sham Glomerular accumulation of CD4ϩ T cells (IL-12 chimeras, chimeras, 4.6 Ϯ 0.1 mg/24 h) and serum creatinine (WT, 35 Ϯ 0.7 Ϯ 0.1 c/gcs; P Ͻ 0.0001) and macrophages (IL-12 chime- 3 ␮mol/L; sham chimeras, 36 Ϯ 6 ␮mol/L). The interstitial ras, 1.7 Ϯ 0.2 c/gcs; P Ͻ 0.0001) was reduced compared with inflammatory injury was equivalent (WT, 141 Ϯ 4.2 cells/ both WT and sham chimeric mice with GN (Figure 3). Pro- mm2; sham chimeras, 147 Ϯ 5.5 cells/mm2; Figure 4). teinuria was reduced in IL-12 chimeras (1.9 Ϯ 0.4 mg/24 h; P However, despite equivalent systemic immune responses to Ͻ 0.01) compared with WT and sham chimera. Serum creat- the nephritogenic antigen to WT and sham chimeric mice, inine levels in IL-12 chimeras with GN (26 Ϯ 1 ␮mol/L; P Ͻ IL-12 chimeric mice developed significantly attenuated cres- 0.05) were significantly reduced compared with WT sham centic GN, indicating that IL-12 from intrinsic renal cells is chimeric mice with GN (Figure 4). The interstitial inflamma- required for full expression of immune renal injury in this tory infiltrate in IL-12 chimeric mice (IL-12 chimeras, 132 Ϯ model. IL-12 staining in the chimeric mice demonstrated lo- 5.0 cells/mm2) was not significantly reduced compared with calization of IL-12 expression on the surface of intraglomerular WT and sham chimeric mice (Figure 4). macrophages and limited expression in areas immediately sur- rounding these macrophages. Periglomerular macrophages did Discussion not express IL-12, and IL-12 expression was not detected in the IL-12 promotes Th1 responses that are protective against tubules (Figure 2E). Histologic appearances of crescentic GN intracellular pathogens such as Leishmania, Listeria, Toxo- were attenuated (Figure 1E), and the incidence of crescentic plasma, and Mycobacteria (24). In murine models of organ- glomeruli was significantly reduced (IL-12 chimeras, 16 Ϯ specific, cell-mediated immune injury such as experimental 0.6%; P Ͻ 0.0001 compared with WT and sham chimeras). allergic encephalomyelitis (25) and autoimmune diabetes (26), J Am Soc Nephrol 12: 464–471, 2001 Intrinsic IL-12 Augments Renal Injury 469

sham chimeric mice with GN. In glomeruli, IL-12 was dem- onstrated on the surface of and adjacent to macrophages. IL-12 expression was also observed in areas of glomeruli remote from macrophages, consistent with production by intrinsic glomerular cells. Mesangial cells have been demonstrated to express IL-12 p40 mRNA and p70 protein in response to proinflammatory stimuli, including tumor necrosis factor-␣ and lipopolysaccharide (19). Periglomerular and interstitial macrophages showed minimal IL-12 expression, suggesting they may have a different activation status and different func- tions to intraglomerular macrophages. In IL-12 chimeric mice, IL-12 expression was restricted to the surface of macrophages and their immediate surrounds. IL-12 expression was not observed in tubules and the intersti- Figure 5. Serum titers of mouse anti-sheep globulin Ab in WT (᭜), sham chimeric (⅜), IL-12 chimeric (●), and IL-12–deficient (᭛) tial inflammatory infiltrate was not attenuated, indicating that mice developing crescentic glomerulonephritis. A titration of normal this infiltrate is not dependent on tubular IL-12 production. mouse serum (f) is shown for comparison. Theses studies provide the first demonstration of an important functional role for a cytokine derived from nonimmune intrin- sic renal cells in immune renal injury. GN in IL-12 chimeric mice was not reduced to the extent seen in IL-12–deficient IL-12 plays an important role by directing injurious Th1 re- mice, indicating that the role of local IL-12 production is sponses. A similar role for IL-12 in facilitating injurious im- complementary to that of bone marrow–derived cell IL-12 mune responses has been demonstrated in cutaneous DTH production. (27). A previous study, in which an anti–IL-12 mAb was used IL-12 from intrinsic renal cells may act in an autocrine or a to functionally inhibit IL-12 in vivo, demonstrated that IL-12 paracrine manner to amplify immune renal injury. Mesangial also promotes Th1 responses and renal injury in murine cres- cells have been demonstrated recently to express the IL-12 centic GN (8). The current study provides confirmation of the receptor and respond to IL-12 in vitro by increasing production important role of IL-12 in cutaneous DTH and crescentic GN of inflammatory mediators, platelet activating factor, and re- by demonstrating marked protection from Ag-specific skin active oxygen species (19), consistent with an autocrine role swelling and immune renal injury in IL-12–deficient mice. for mesangial cell–derived IL-12 in crescentic GN. Paracrine IL-12 production by nonimmune cells has been demon- effects of IL-12 may be exerted on intraglomerular T cells and strated in a variety of organs, including the kidney (6,18) and macrophages or endothelial cells. The potential for IL-12 to skin (28,29). However, the contribution of IL-12 production by increase expression of intracellular adhesion molecule 1 or these nonimmune cells to organ-specific immune injury is E-selectin (30) or to increase production of and unknown. This issue was addressed in a model of immune RANTES has been demonstrated in other diseases (31,32). renal injury by using IL-12 chimeric mice, created by trans- Intrinsic renal cell expression of major histocompatibility plantation of normal bone marrow cells in to IL-12–deficient complex II was demonstrated recently to be required for renal mice. These chimeric mice had normal circulating leukocytes recruitment of T cells in this model of crescentic GN (33), and and lymphocyte subsets and normal splenic architecture with simultaneous expression of IL-12 by these intrinsic cells may regard to CD4ϩ T-cell and macrophage distribution. Their augment Th1 activation and proinflammatory effector func- baseline proteinuria, serum creatinine, and renal histology were tions of these T cells after recruitment. IL-12 has been shown normal, indicating no protracted effects on the kidney, 8 wk to promote Th1 development, stimulate Th1 cell proliferation, after irradiation. Serum Ab titers and cutaneous DTH to the and induce IFN-␥ production by resting and activated T cells nephritogenic Ag were not different, indicating similar sys- (34–36). IFN-␥ stimulates the release of chemotac- temic immune responses in each group. The observation that tic protein-1 (37) and production of proinflammatory cyto- antigen-specific skin swelling was not affected in IL-12 chi- kines, including IL-12 (16). IL-12–deficient mice have defi- meric mice indicates that IL-12 expression by non–bone mar- cient IFN-␥ production (20), and IFN-␥ has been demonstrated row–derived cells in the skin, e.g., keratinocytes, is not re- to be an important proinflammatory mediator in crescentic GN quired for development of cutaneous DTH. This is consistent (4,7). However, one report suggests that the absence of the with the view that the major effector cells of cutaneous DTH IFN-␥ receptor does not alter crescent formation in anti-GBM are bone marrow–derived Langerhans cells, CD4ϩ T cells, and nephritis (38). macrophages. In summary, these studies demonstrate that IL-12 production Crescent formation, glomerular inflammatory cell influx, by non–bone marrow–derived cells is required for the full and functional renal injury were significantly reduced, indicat- expression of a Th1-dependent model of immune renal injury ing that IL-12 from intrinsic renal cells makes a significant but not for cutaneous DTH. They provide the first demonstra- contribution to the effector phase of this disease. IL-12 expres- tion of the capacity of a cytokine derived from intrinsic renal sion was demonstrated in glomeruli and tubules of WT and cells to augment immune renal injury and suggest that IL-12 470 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 464–471, 2001 production by intrinsic renal cells, demonstrated in vitro and in production by monocytes via CD40-CD40 ligand interaction. human GN (39), may be an important modulator of renal Eur J Immunol 25: 1125–1128, 1995 injury. 15. Manetti R, Parronchi P, Giudizi MG, Piccinni MP, Maggi E, Trinchieri G, Romagnani S: stimulatory factor (interleukin 12 [IL-12]) induces T helper type 1 (Th1)-specific Acknowledgments immune responses and inhibits the development of IL-4-produc- These studies were supported by a grant from the National Health ing Th cells. J Exp Med 177: 1199–1204, 1993 and Medical Research Council of Australia (NH&MRC). Dr, Tipping 16. Trinchieri G: Interleukin-12: A proinflammatory cytokine with is an NH&MRC Senior Research Fellow. We thank J. Sharkey, A. immunoregulatory functions that bridge innate resistance and Wright, D. Calleci, and T. Niklou for their technical assistance and Dr. antigen-specific adaptive immunity. Annu Rev Immunol 13: 251– G. Trinchieri for the anti–IL-12 mAb (clone 15.6). 276, 1995 17. Chehimi J, Trinchieri G: Interleukin-12: A bridge between innate resistance and adaptive immunity with a role in infection and References acquired immunodeficiency. 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