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J Am Soc Nephrol 14:2813-2822, 2003
© 2003 American Society of Nephrology

BASIC SCIENCE

Intrinsic Renal Cell Expression of CD40 Directs Th1 Effectors Inducing Experimental Crescentic Glomerulonephritis

Amanda-Jane Ruth, A. Richard Kitching, Timothy J. Semple, Peter G. Tipping and Stephen R. Holdsworth

Centre for Inflammatory Diseases, Monash University, Department of Medicine, Monash Medical Centre, Clayton, Victoria, Australia.

Correspondence to Dr. Stephen R. Holdsworth, Department of Medicine, Monash University, Monash Medical Centre, Level 5, Block E, 246 Clayton Rd., Clayton, Victoria 3168, Australia. Phone: 61-3-9594-5525; Fax: 61-3-9594-6437;


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Evidence suggests that human and experimental crescentic GN results from Th1-predominant immunity to glomerular antigens. CD40/CD154 signaling plays a key role in initiating Th1 responses and may direct Th1 effector responses. The role of CD40 in the development of GN was assessed in murine experimental anti-glomerular basement membrane GN. In this model, C57BL/6 wild-type (WT) mice sensitized to sheep globulin develop crescentic GN resulting from Th1 effector responses when challenged with sheep globulin planted in glomeruli. CD40-/- mice do not develop immunity in response to sheep globulin and thus fail to develop effector responses or significant GN. CD40 is expressed in nephritic glomeruli, suggesting a potential role for intrarenal CD40-CD154 interactions in injurious effector responses. Immune neutralization of the CD40 ligand (CD154) at the time of challenge significantly reduced accumulation of Th1 effectors and injury. The role of CD40 expression by renal cells was assessed by comparing GN in WT->CD40-/- chimeras (absent renal but intact bone marrow CD40) and sham chimeric mice (WT->WT). Both groups developed strong antigen-specific immune responses (antibody and IFN-{gamma} production). However, WT->CD40-/- chimeras demonstrated reduced renal monocyte chemotactic protein 1 and IFN-inducible protein 10 mRNA levels and minimal T cell and macrophage influx and were protected from renal injury. Sham chimeric mice developed reduced GFR, with prominent renal expression of monocyte chemotactic protein 1 and IFN-inducible protein 10 mRNA and effector cell accumulation. In conclusion, the expression of CD40 by nonimmune renal cells plays a major role in Th1 effector responses by inducing Th1 chemokine production. Therefore, CD40-CD154 interactions are a potential therapeutic target in GN. E-mail: Stephen.Holdsworth@med.monash.edu.au


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cell-cell interactions play a vital role in mediating adaptive immune responses. Two types of signals have been defined as being required for the initiation of T cell-dependent immune responses (1). The first type is the T cell receptor/MHC-mediated signal and the second type results from the binding of costimulatory/accessory receptor ligand pairs, which bidirectionally deliver signals to the T cells and antigen-presenting cells to confer functional activity (2). CD40/CD154 is one such costimulatory pair whose regulatory role in the initiation of adaptive immune responses has been clearly defined (3). The importance of CD40/CD154 signaling in the mediation of effector responses has also been highlighted. The CD40 signal has been demonstrated to be required for delivery of optimal functional capacity to macrophages, with increases in the production of IL-12, IL-8, and IL-1 (4,5). Ligation of CD40 expressed by macrophages leads to upregulation of intercellular adhesion molecule-1, MHC class II, and B7-2 (6). CD40-dependent activation of endothelial cells leads to upregulation of adhesion molecules, facilitating leukocyte migration to inflammatory sites (7,8). The CD40 signal enhances effector functions in concert with the production of soluble factors and other costimulatory signals.

Crescentic GN is the most injurious form of GN, with the worst outcomes. Patients with the most severe cases of crescentic GN present with hypercellular glomeruli and reduced GFR, which frequently progresses to renal failure. Evidence from studies of experimental crescentic GN suggest that glomerular injury results from Th1-predominant nephritogenic immune responses to antigens deposited or endogenously expressed in glomeruli (9). Observations in human crescentic GN also demonstrate the prominent participation of delayed-type hypersensitivity (DTH) effectors (10), suggesting that injury in this setting results from Th1-predominant nephritogenic immunity. Renal injury in crescentic GN results from DTH-like responses that lead to the accumulation of CD4+ T cells (11) and macrophages (12) in glomeruli. Recruitment of effector cells is directed by cell-cell interactions that result in the production of chemokines and adhesion molecules (13). Chemokines known to recruit Th1 effectors and macrophages, including IFN-inducible protein 10 (IP-10) and monocyte chemotactic protein 1 (MCP-1), have been demonstrated in biopsies from patients with crescentic GN (14,15).

CD154 is predominantly expressed on activated T cells (16). CD40 expression is not confined to immune cells, however, but is upregulated in inflamed tissue. For example, CD40 has been demonstrated to be expressed by parenchymal microglial cells in experimental autoimmune encephalomyelitis (17) and by myocardial cells in murine viral myocarditis (18). Renal biopsies from patients with proliferative GN demonstrated upregulated CD40 expression in mesangial cells, endothelial cells, and distal and proximal tubules (19). CD40 expression on renal tubular epithelial cells was demonstrated to be upregulated in the presence of IL-1 in vitro (20). Cell-cell interactions are likely to be important at a local effector level. Immune cells were previously demonstrated to upregulate inflammatory responses through CD40-dependent interactions with resident renal cells in vitro, with enhanced IL-6, MCP-1, and intercellular adhesion molecule-1 production (21). The production of IP-10, regulated upon activation, normal T cell expressed and secreted (RANTES), and MCP-1 is upregulated upon the interaction of intrinsic renal cells with activated T cells. In that system, RANTES production was demonstrated to be dependent on CD154 in vitro (22). These chemokines are largely involved in the recruitment of macrophages and T cells (23). These observations suggest a potential role for CD40 expression by inflamed renal cells in the development of Th1 effector responses in glomeruli, although the molecular processes remain to be defined.

We sought to investigate the roles of CD40 and CD154 in mediating the development of GN. We used a murine model of GN that closely resembles human nephritis, with crescent formation, renal failure, and prominent DTH effectors directed by Th1 responses to a planted antigen (9). CD40 expression by nephritogenic glomeruli was prominent, which suggested a functional role for CD40-CD154 interactions in the effector phase of disease. The use of an inhibitory antibody to CD154 confirmed the requirement for this interaction in the injurious effector response. Generation of chimeric mice with absent CD40 in peripheral target tissues (glomeruli) but intact leukocyte CD40 expression and systemic immunity allowed evaluation of the role of CD40 expression by intrinsic renal cells in the effector phase of this disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Breeding pairs of mice with targeted disruption of the CD40 gene (24) were obtained from The Jackson Laboratory (Bar Harbor, ME). The mice have been backcrossed to a C57BL/6 background for nine generations. Mice were housed and bred under specific pathogen-free conditions (Monash University, Clayton, Australia).

Induction of Accelerated Experimental Anti-Glomerular Basement Membrane GN
Sheep anti-mouse glomerular basement membrane (GBM) globulin was prepared as described previously (25). Accelerated experimental anti-GBM GN was induced by sensitizing 8- to 10-wk-old male mice with subcutaneous injections, in each flank, of a total of 500 µg of sheep globulin in 100 µl of Freund’s complete adjuvant. GN was initiated 10 d later by intravenous challenge with 12 mg of sheep anti-mouse GBM globulin. Immune responses and renal injury were assessed 10 d later, in CD40-/- (n = 6) and wild-type (WT) (n = 6) mice.

Treatment with Anti-CD40 Ligand mAb
Six hours before intravenous challenge with anti-GBM globulin, C57BL/6 mice (n = 6) were treated intraperitoneally with 1 mg of hamster anti-mouse CD40 ligand mAb (MR1; Taconic Biotechnology, Germantown, NY); the mice received 250 µg intraperitoneally every 2 d thereafter. WT control IgG-treated mice (n = 6) were treated with guinea pig IgG (Sigma-Aldrich, St. Louis, MO).

Bone Marrow Transplantation
CD40-/- (n = 6) and WT (n = 6) recipient mice (age, 5 wk) received 1100 rad of total-body irradiation. Bone marrow (BM) cells were harvested aseptically from the femurs and tibias of WT donor mice. Recipient mice were injected with 5 x 106 leukocytes within 24 h after irradiation. BM reconstitution occurred in a period of 8 wk, during which the mice were housed under specific pathogen-free conditions (26). CD40-/- mice transplanted with WT BM (WT->CD40-/-) and sham chimeric mice (WT->WT) were used for the induction of nonaccelerated experimental anti-GBM GN with the intravenous administration of 20 mg of sheep anti-mouse GBM globulin. Immune responses and renal injury were assessed 21 d after injection.

Assessment of Glomerular Injury
Glomerular Crescent Formation and Hypercellularity.
Kidney tissue was fixed in Bouin’s fixative and embedded in paraffin, and 4-µm tissue sections were cut and stained with periodic acid-Schiff reagent. Glomerular crescent formation was assessed with a blinded protocol in which at least 25 glomeruli were assessed to determine the crescent score for each animal. Crescent formation was considered to be apparent when glomeruli exhibited more than two layers of cells in Bowman’s space. Glomerular cellularity was assessed by counting the number of nuclei in 20 glomeruli for each animal and was expressed as cells per glomerular cross-section.

GFR.
Creatinine clearance was assessed to determine GFR. Urine was collected during the 24-h period before euthanasia, with the aid of metabolic cages. Serum was collected from blood obtained on the day of euthanasia. Serum and urine creatinine concentrations were measured with an enzymatic creatininase method (CREA Plus, catalog no. 1775685; Boehringer Mannheim, Indianapolis, IN), using a Roche Cobas bioanalyzer (Roche, Nutley, NJ).

Glomerular CD4+ T Cell and Macrophage Accumulation
Periodate/lysine/paraformaldehyde-fixed kidney sections (6 µm) were stained to demonstrate CD4+ T cells and macrophages, with a three-layer immunoperoxidase technique (27). The primary mAb used were GK1.5 (anti-mouse CD4; American Type Culture Collection, Manassas, VA) and M/170 (anti-mouse Mac-1; American Type Culture Collection). At least 20 glomeruli were assessed for each animal, and results were expressed as cells per glomerular cross-section.

CD40 Expression
Acetone-fixed kidney sections (6 µm) were stained to demonstrate CD40 expression, with a three-layer immunoperoxidase technique. The primary mAb used was rat anti-mouse CD40 (clone 3/23; BD Biosciences, North Ryde, Australia), and the secondary antibody used was rabbit anti-rat biotin (BD Biosciences). Endogenous biotin was blocked by using a biotin-blocking system (Dako, Botany, Australia), and CD40 was detected by using an avidin-biotin complex detection system (Dako). Kidney sections from WT mice with GN, normal mice, WT->CD40-/- chimeras, and sham chimeras (WT->WT) were assessed for CD40 expression.

Assessment of Systemic Immune Responses
Humoral Responses to Sheep Globulin.
Mouse anti-sheep globulin antibody titers in sera were measured at the end of each experiment with an ELISA, as described previously (28).

Induction of Cutaneous DTH.
Mice with experimental anti-GBM GN were challenged with an intradermal injection, into the right plantar footpad, of 400 µg of sheep globulin in 40 µl of PBS. The same dose of an irrelevant antigen (horse globulin) was injected into the contralateral footpad. Swelling was measured after 24 h, with a micrometer (Mitutoyo, Kawasaki-shi, Japan). DTH was assessed as the measured difference in swelling between the two footpads and was expressed in millimeters.

Assessment of Splenocyte IFN-{gamma} Production
Spleens were aseptically removed from WT->CD40-/- and sham chimeric mice 21 d after injection of anti-GBM globulin. Single-cell suspensions were prepared in DMEM/5% FCS (28). Splenocytes (4 x 106 splenocytes/ml, in DMEM/10% FCS) were incubated for 72 h at 37°C with protein G-purified normal sheep IgG (10 µg/ml). IFN-{gamma} levels in the supernatant were measured with an ELISA, as described previously (28). The following mAb were used for the IFN-{gamma} ELISA: rat anti-mouse IFN-{gamma} (RA-6A2; Pharmingen, San Diego, CA) and biotinylated rat anti-mouse IFN-{gamma} (XMG1.2; Pharmingen).

RNase Protection Assays
RNA Extraction.
Total-kidney RNA was extracted with TRIzol reagent (Life Technologies BRL, Mount Waverley, Australia), according to the manufacturer’s protocol. The final product was air-dried, dissolved in ultrapure, DNase/RNase-free water (Life Technologies), and stored at -80°C. RNA concentrations were determined with spectroscopic assessments at 260 nm.

RNase Protection Assay.
Total-kidney RNA was assessed by using the RiboQuant system (Pharmingen). Multiprobes incorporating [{alpha}-32P]UTP were transcribed from the template set mCK-5c with T7 RNA polymerase in vitro transcription. After DNase I treatment, the riboprobes were isolated by phenol/chloroform extraction and precipitation with 4 M ammonium acetate and ethanol. The incorporation of [{alpha}-32P]UTP was determined by assessment of Cherenkov activity in a scintillation counter. The probe was diluted to 3.5 x 105 cpm/µl and then added to 20 µg of total-kidney RNA. Hybridization and isolation were performed according to the protocol in the RiboQuant manual. RNA hybrids were separated by electrophoresis on a 5% polyacrylamide/8 M urea gel. The gel was dried at 80°C for 1 h before being exposed to the imaging plate of a FLA-2000 phosphoimager (Fuji Photo Film Co., Tokyo, Japan). Image Gauge software (version 3.46; Fuji Photo Film Co.) was used to evaluate the gel image. Chemokine expression was measured and normalized to the housekeeping gene L32.

Statistical Analyses
Mann-Whitney U tests were used to analyze differences for all parameters examined. Statistical significance was defined as P < 0.05. Data were expressed as means ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD40-/- Mice Do Not Produce Nephritogenic Immune Responses and Consequently Fail to Develop Experimental Crescentic GN
CD40-intact C57BL/6 (WT) mice immunized with sheep globulin developed humoral immunity and dermal DTH (0.13 ± 0.02 mm, P < 0.002) in response to the nephritogenic antigen (Figure 1). Ten days after glomerular antigen challenge with sheep anti-mouse GBM globulin, CD40-intact mice demonstrated prominent glomerular CD40 expression. Few CD40-positive interstitial cells were observed, and tubular epithelial cells of diseased kidneys did not exhibit CD40 expression (Figure 2). Accumulation of Th1 effectors, namely, CD4+ T cells (1.6 ± 0.2 cells/glomerular cross-section) and macrophages (2.0 ± 0.2 cells/glomerular cross-section), in the glomeruli of WT mice was observed, resulting in proliferative crescentic GN (crescent formation, 41 ± 4%) (Figure 3).



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  		Figure 1. Systemic immune responses in wild-type (WT) ({blacktriangleup}) and CD40-/- ({blacksquare}) mice with experimental anti-glomerular basement membrane (GBM) GN. (A) WT mice produced significant circulating antibody titers (P < 0.0001), whereas CD40-/- mice exhibited minimal antibody responses. (B) WT mice developed cutaneous delayed-type hypersensitivity (DTH) after antigen challenge, whereas responses in CD40-/- mice were markedly attenuated (*P < 0.002).

 


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  		Figure 2. Immunohistologic staining for glomerular CD40 expression. CD40 was not expressed in normal glomeruli (A), but diffuse staining was prominent in the glomeruli of WT mice with GN (B). Glomeruli from WT->CD40-/- chimeric mice displayed few intraglomerular cells expressing CD40 (arrows) (C). A pattern of expression similar to that of WT mice was observed in the glomeruli of sham chimeric mice (D).

 


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  		Figure 3. Assessment of glomerular DTH effectors in experimental GN. Glomeruli of WT mice exhibited prominent CD4+ T cell (A) and macrophage (B) influx and crescent formation (C). The development of glomerular crescents and T cell and macrophage accumulation were all significantly less in CD40-/- mice (*P < 0.005). Dotted lines, normal values. c/gcs, cells/glomerular cross-section.

 
CD40-/- mice failed to develop significant immunity in response to sheep globulin, as demonstrated by the absence of humoral antibody production and skin DTH responses (Figure 1). Consequently, glomerular Th1 effector cell accumulation (CD4+ T cell and macrophage glomerular infiltration) was absent from the glomeruli of CD40-/- mice after antigen challenge (Figure 3). Only mild histologic injury was apparent (crescent formation, 9 ± 3%; P < 0.0005, compared with control values).

Blockade of the CD40/CD154 Signal in the Effector Phase Prevents the Accumulation of Th1 Effectors and the Development of Crescentic GN
Titers of circulating anti-sheep globulin antibody were similar in control and anti-CD154-treated mice after immunization with the nephritogenic antigen (Figure 4A), suggesting that late inhibition of the CD40/CD154 signal did not affect established immune responses to the antigen. Dermal DTH was prevented in anti-CD154-treated mice (0.03 ± 0.013 mm), in contrast to control IgG-treated animals (0.23 ± 0.08 mm, P < 0.001) (Figure 4B). Blockade of the CD40 signal in the effector phase reduced the accumulation of DTH effectors at the site of antigen challenge (CD4+ T cells: 0.4 ± 0.1 cells/glomerular cross-section; control, 1.9 ± 0.2 cells/glomerular cross-section; P < 0.02; macrophages: 0.7 ± 0.2 cells/glomerular cross-section; control, 3.5 ± 0.7 cells/glomerular cross-section; P < 0.02) (Figure 5, A and B). Therefore, CD40 blockade led to a marked reduction in renal injury, as indicated by significantly decreased crescent formation (14 ± 3%; control IgG-treated mice, 29 ± 2%; P < 0.003) (Figure 5C).



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  		Figure 4. Characterization of the systemic immune response of mice treated with anti-CD154 in the effector phase of experimental anti-GBM GN. (A) Anti-mouse CD154-treated ({blacksquare}) and control IgG-treated ({blacktriangleup}) mice developed similar antibody responses to sheep globulin. (B) Cutaneous DTH responses to the nephritogenic antigen were attenuated in mice treated with inhibitory anti-CD154, compared with control IgG-treated animals (*P < 0.05).

 


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  		Figure 5. Accumulation of DTH effectors and assessment of renal injury in anti-CD154-treated mice. Significant reductions in the accumulation of CD4+ T cells (*P < 0.02) (A) and macrophages (*P < 0.02) (B) and in glomerular crescent formation (*P < 0.003) (C) were evident in the glomeruli of mice treated with an inhibitory antibody to CD154, compared with control IgG-treated mice. Dotted lines, normal values. c/gcs, cells/glomerular cross-section.

 
Nephritogenic Immune Responses Develop to Similar Degrees in WT->CD40-/- Chimeras and Sham Chimeric Mice
Similar immune responses were observed in WT->CD40-/- and WT->WT chimeric mice, as demonstrated by similar circulating antibody levels (Figure 6A) and dermal DTH responses (0.11 ± 0.03 mm; sham, 0.12 ± 0.02 mm; P = 0.66) (Figure 6B). However, IFN-{gamma} production by antigen-stimulated splenocytes was significantly higher in WT->CD40-/- chimeras (248 ± 45 pg/105 cells per 72 h), compared with WT->WT chimeric mice (105 ± 13 pg/105 cells per 72 h, P < 0.05) (Figure 6C).



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  		Figure 6. Restoration of systemic immune responses in WT->CD40-/- chimeric mice. WT->WT chimeras exhibited similar antibody (A) and DTH (B) responses to sheep globulin, compared with WT->WT mice (P = 0.66). IFN-{gamma} production by antigen-stimulated splenocytes was greater in WT->CD40-/- chimeras (C), compared with WT->WT chimeras (*P < 0.05).

 
CD40 Is Prominently Expressed in Nephritic Glomeruli of Sham Chimeric Mice Developing GN but Is Absent in the Kidneys of WT->CD40-/- Chimeric C57BL/6 Mice
CD40 expression was markedly reduced in glomeruli of WT->CD40-/- chimeric mice developing GN. Occasional positive cells (similar to macrophage influx) were observed in glomeruli (Figure 2C). The expression of CD40 in WT->WT chimeric mice with GN was similar to that observed in WT mice with GN, with a diffuse intraglomerular pattern of staining (Figure 2D).

Intrinsic Renal Cell CD40 Signaling Plays a Vital Role in Directing Glomerular Effectors of DTH
In the absence of renal CD40, glomerular accumulation of DTH effectors, namely, CD4+ T cells (0.6 ± 0.3 cells/glomerular cross-section) and macrophages (2.1 ± 0.3 cells/glomerular cross-section), occurred at significantly reduced levels, compared with the high degree of cellular infiltration observed in the glomeruli of sham chimeric mice (CD4+ T cells, 2.1 ± 0.51 cells/glomerular cross-section, P < 0.001; macrophages, 5.2 ± 0.6 cells/glomerular cross-section, P < 0.005) with CD40-intact BM-derived cells (Figure 7).



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  		Figure 7. Accumulation of glomerular DTH effectors in WT->CD40-/- mice with GN. CD4+ T cell influx was abrogated (A) and macrophage accumulation was significantly reduced (*P < 0.001) (B), compared with WT->WT chimeras. Dotted lines, normal values. c/gcs, cells/glomerular cross-section.

 
Renal Injury Is Significantly Diminished in WT->CD40-/- Chimeric Mice
Renal injury in sham chimeric mice was associated with reduced GFR (102 ± 11 µl/min) (Figure 8A). In contrast, WT->CD40-/- chimeras exhibited GFR within the normal range (155 ± 16 µl/min, P < 0.05, compared with control values). Histologic cellular proliferation was abnormal in sham chimeras with GN (glomerular cellularity, 43.9 ± 1.5 cells/glomerular cross-section) and moderate crescent formation (13 ± 2%); however, WT->CD40-/- chimeras displayed normal glomerular cellularity (28.4 ± 0.8 cells/glomerular cross-section, P < 0.005) and minimal crescent formation (3 ± 2%, P < 0.005, compared with sham chimeras) (Figure 8B).



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  		Figure 8. Glomerular injury in WT->CD40-/- chimeric mice. Glomerular injury did not develop in the absence of intrinsic renal CD40 expression. WT->CD40-/- chimeric mice with GN exhibited normal GFR (A) and cellularity (B). Conversely, WT->WT chimeric mice exhibited reduced creatinine clearance (*P < 0.05) and developed hypercellularity (*P < 0.005). Dotted line, normal values.

 
Glomerular Expression of Chemokine mRNA Is Reduced in WT->CD40-/- Chimeras
Renal expression of the chemokines IP-10, MCP-1, and RANTES was upregulated in sham chimeric mice with GN, compared with levels in normal mice (P < 0.05). WT->CD40-/- chimeras exhibited significantly lower (P < 0.001) renal chemokine expression of IP-10 and MCP-1 mRNA, compared with sham chimeras that possessed intact CD40-expressing cells (Figure 9).



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  		Figure 9. Renal chemokine expression assessed on the basis of mRNA levels, relative to levels of the housekeeping gene L32. WT->CD40-/- chimeric mice exhibited significantly lower renal expression of IFN-inducible protein 10 (IP-10) (A) and monocyte chemotactic protein 1 (MCP-1) (B) mRNA, compared with WT->WT chimeras (*P < 0.05). Regulated upon activation, normal T cell expressed and secreted (RANTES) expression was unaffected by the absence of intrinsic renal CD40 (C). Dotted lines, values in normal mice. AU, absorbance units.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These experiments used a murine model of anti-GBM GN that closely resembles crescentic GN in human subjects (9). Human crescentic GN is characterized by prominent accumulation of Th1 effectors and proliferation of intrinsic glomerular cells. The murine model of GN results from Th1-polarized nephritogenic immune responses to an exogenous antigen (sheep globulin), which induce glomerular DTH effector responses when the immunogen is targeted to glomeruli as sheep anti-mouse GBM globulin.

CD40 and CD154 play essential roles in the initiation of Th1 responses (29). It is not surprising that CD40-/- animals do not develop GN, because these mice are incapable of mounting Th1 immune responses to the nephritogenic antigen. The roles of CD40 and CD154 in effector responses have been less well characterized but are clinically relevant, because patients with GN present with established nephritogenic immunity. The use of an accelerated model of experimental anti-GBM GN provides clear definition of the onset of the effector response. Injury is induced by the challenge of sensitized animals with sheep anti-mouse GBM globulin. Experimental crescentic GN results from Th1 effector responses to glomerular antigens (11). Administration of the inhibitory mAb to CD154 abrogated glomerular influx of CD4+ T cells and macrophages. The reductions in the accumulation of those effectors were associated with protection from the development of injury, despite the maintenance of serum anti-sheep globulin antibody levels, which is consistent with the requirement for glomerular Th1-directed DTH in the mediation of injury in crescentic GN (30). The absence of skin DTH in anti-CD154-treated animals is likely the result of inhibition of CD40/CD154-mediated effector responses and confirms a role for CD40/CD154-mediated Th1 effector responses in different target organs. Collectively, the results demonstrate that CD40-CD154 interactions have a distinct role in effector responses, in addition to their known function of initiating immune responses.

The prevention of disease development has been demonstrated in a variety of animal models, including graft versus host disease (31), experimental allergic encephalomyelitis (32), collagen-induced arthritis (33), and autoimmune thyroiditis (34), when animals are treated with inhibitory anti-CD154 antibodies at the onset or in the early stages of disease initiation. Blockade of the CD40/CD154 signal has been demonstrated to decrease inflammation in models of antibody-mediated membranous GN when animals are treated in the early stages of disease development (35) and in murine models of autoimmune lupus nephritis (36,37). However, the effect of CD40/CD154 inhibition on effector responses is difficult to separate from the potential effect on the development of autoimmunity in those models.

CD40 and CD154 play roles in cutaneous inflammation through antigen-presenting cell/cognate CD4+ T cell antigen presentation and recognition in the skin and through IFN-{gamma}-induced upregulation of CD40 on keratinocytes, which contribute to the recruitment of T cells and macrophages through the production of soluble chemoattractant factors (38). These studies provide evidence that CD40 expression by intrinsic renal cells may serve a role in GN similar to that of tissue-expressed CD40 in the skin. Our data suggest an additional role for CD40 interactions in effector responses within glomeruli in the presence of systemic immunity. CD40 is expressed by nonimmune cells in renal disease. Immunohistochemical analyses of WT mice with GN demonstrated strong intraglomerular CD40 expression, which was not present in normal animals. CD40 was expressed in a pattern that was not confined to immune cells, suggesting upregulation by intrinsic renal cells. A similar distribution of CD40 expression was observed in glomerular crescents of renal biopsy specimens obtained from patients with World Health Organization class IV lupus nephritis (19).

Because of the requirement for CD40/CD154 signaling in the mediation of Th1 effector responses, we explored a role for renal CD40 signals in mediating effector responses and disease development. BM CD40-intact mice with CD40-deficient peripheral tissues (WT->CD40-/- chimeras) were created with an irradiation/BM transplantation technique that has been successfully used to reconstitute mice with genotypically distinct BM-derived cells (39). Immune responses were similar in WT->CD40-/- mice and sham chimeras (WT->WT), which displayed similar levels of humoral antibody production and cutaneous DTH, confirming reconstitution of intact circulating T and B cell compartments in those animals. IFN-{gamma} production by antigen-stimulated splenocytes from WT->CD40-/- chimeras was greater than that by cells from sham chimeric mice. Increased IFN-{gamma} production by WT->CD40-/- chimeras suggests that CD40 expression by parenchymal splenic cells downregulates IFN-{gamma} production by antigen-stimulated splenocytes in vitro. Because IFN-{gamma}-dependent cutaneous DTH responses to the same antigen are equivalent in WT->CD40-/- and sham chimeric mice, these in vitro differences in IFN-{gamma} production do not seem to result in significant functional consequences in vivo. Humoral immune responses to the nephritogenic antigen were also similar in WT->CD40-/- and sham chimeras. We previously demonstrated that increased IFN-{gamma} production is associated with the development of Th1 responses, leading to GN (11,25). The presence of similar systemic immunity in response to sheep globulin indicates that similar levels of renal injury would be expected to develop in WT->CD40-/- chimeras and sham chimeric mice after glomerular antigen challenge. However, in the absence of renal CD40, glomerular antigen challenge produced no significant T cell infiltrate and reduced macrophage accumulation. The successful peripheral deletion of CD40 in chimeric mice was confirmed by the absence of intrinsic renal CD40 expression in the glomeruli of WT->CD40-/- chimeras and the demonstration of CD40 in the glomeruli of sham chimeric mice with GN. A prominent influx of CD4+ T cells and macrophages occurred in glomeruli of sham chimeric mice and was associated with glomerular hypercellularity, crescent formation, and significantly reduced GFR (creatinine clearance). WT->CD40-/- chimeric mice demonstrated minimal hypercellularity and crescent formation and were completely protected from renal failure in this model.

We previously demonstrated that renal injury in this model is dependent on Th1-directed DTH and is independent of circulating antibody (30). Therefore, renal expression of CD40 is essential for Th1 effector accumulation and DTH-mediated injury. The development of cutaneous DTH in WT->CD40-/- chimeras reflects the fact that the skin (unlike the kidney) (39) is richly endowed with defensive, resident, professional, BM-derived, immune cells (capable of expressing CD40), which are repopulated during the 3 mo of BM reconstitution in this model.

Our data demonstrate a requirement for renal CD40 expression in mediation of the accumulation of DTH effectors in glomeruli, the development of renal injury, and local renal production of the chemokines IP-10 and MCP-1. Previous studies demonstrated that renal tubular epithelial cells represent a source of MCP-1, IP-10, and RANTES (22) and that RANTES is produced through CD40 ligand-dependent mechanisms (20). Chemokine production by nonimmune cells also occurs in experimental autoimmune encephalomyelitis and is regulated by CD40-bearing parenchymal cells (40) and by myoblasts in inflamed muscle (41). WT->CD40-/- chimeras exhibited little Th1 cell glomerular accumulation and significantly reduced renal IP-10 expression. Therefore, the engagement of CD4+ T cells expressing CD154 with CD40 on intrinsic renal cells may enhance local IP-10 production.

We previously demonstrated that intrinsic renal cell MHC class II expression facilitates glomerular DTH through the recruitment of Th1 cells (42). Renal cells that have the capacity to present antigen may deliver signals to T cells via MHC class II peptide recognition, in concert with CD40/CD154 ligation. Therefore, renal CD40/CD154 effector T cell engagement may provide a second signal facilitating effector cell accumulation in glomeruli.

Macrophages have been demonstrated to be the major effector cells required for the development of renal injury in crescentic GN (43). Their accumulation is Th1 cell dependent (12). Despite the presence of CD40-expressing macrophages in WT->CD40-/- chimeric mice, reduced accumulation was observed in nephritogenic glomeruli in the absence of renal CD40. This is likely to be attributable to reduced MCP-1 expression in the absence of intrinsic renal cell CD40/CD154 signaling. CD154 expressed by Th1 cells may direct CD40-expressing intrinsic renal cells to facilitate glomerular DTH responses through enhanced local production of MCP-1.

In conclusion, these studies provide evidence that CD40-CD154 interactions are required for the development of GN resulting from Th1 immune responses mediating glomerular DTH. These studies have highlighted the requirement for CD40 and CD40 ligand in the development of DTH effector responses in glomeruli. Studies with WT->CD40-/- chimeras demonstrated that intrinsic renal cell CD40 expression mediates this effect through the accumulation of effectors in glomeruli and the production of renal chemokines, facilitating the progression of renal injury. Therefore, targeting of CD40 and CD154 may have therapeutic relevance for crescentic GN in human subjects.


   Acknowledgments
 
This work was supported by Grant 013201 from the National Health and Medical Research Council of Australia.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication May 23, 2003. Accepted for publication July 10, 2003.




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