Major Histocompatibility Complex Class II Expression by Intrinsic
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Published August 3, 1998 Brief Definitive Report Major Histocompatibility Complex Class II Expression by Intrinsic Renal Cells Is Required for Crescentic Glomerulonephritis By Shuo Li,* Christian Kurts,‡ Frank Köntgen,‡ Stephen R. Holdsworth,* and Peter G. Tipping* From the *Centre for Inflammatory Diseases, Monash University Department of Medicine, Monash Medical Centre, Clayton, 3168, Victoria, Australia; and the ‡Immunology Division, Walter and Eliza Hall Institute, Parkville, 3050, Victoria, Australia Summary The requirement for major histocompatibility complex class II (MHC II) to initiate immune renal injury was studied in a murine model of CD41 T cell–dependent crescentic glomerulo- nephritis (GN). C57BL/6 (MHC II1/1) mice developed crescentic GN with glomerular CD41 T cell infiltration and renal injury, in response to a nephritogenic antigen (sheep globulin) planted on their glomerular basement membrane. MHC II–deficient C57BL/6 mice (MHC II2/2) did not develop crescentic GN, CD41 T cell infiltration, or injury, indicating that this Downloaded from form of immune glomerular injury is MHC II dependent. The requirement for MHC II ex- pression by intrinsic renal cells was studied in chimeric mice, which expressed MHC II on bone marrow–derived cells and in the thymus, but not in the kidneys. These chimeric mice had normal T and B cell populations and MHC II expression in their spleens and lymph nodes and developed an immune response to systemically and cutaneously administered sheep globu- lin. However, they did not develop crescentic GN, CD41 T cell infiltration, or renal injury in response to the sheep globulin planted in their glomeruli. These studies demonstrate that inter- on April 9, 2017 action of CD41 T cells with intrinsic renal cells expressing MHC II is required for develop- ment of cell-mediated immune renal injury. Key words: major histocompatibility complex • glomerulonephritis • T lymphocyte • kidney • mice cell responses are normally dependent on recognition in many organs, including mesangial cells (1, 2) and proxi- Tof antigen bound to MHC molecules on the surface of mal tubular cells in the kidney (3, 4). Thyroid epithelial APCs. MHC class I molecules are predominantly associated cells expressing “aberrant” MHC II can present viral anti- with CD81 cytotoxic T cell responses. MHC class II (MHC gens to cloned human T cells in vitro (5). II) molecules present antigens to CD41 T cells, which stim- Glomerular antibody deposition and/or accumulation of T ulate antibody production and isotype switching and act as cells is observed in the majority of cases of human glomerulo- effector cells for local cell-mediated immune responses. nephritis (GN). These immune effectors localize in response “Professional” APCs are bone marrow–derived cells that to endogenous glomerular antigens or antigens deposited in present antigen to naive T cells in secondary lymphoid tis- glomeruli as components of immune complexes or because of sues. After activation, T cells enter nonlymphoid tissues, their size or charge characteristics. In the most severe forms of where they can recognize antigen and initiate local im- human GN, glomerular injury is manifested by a “prolifera- mune responses. It is well accepted that MHC II is required tive” histological pattern, accumulation of T cells and mac- during activation of naive CD41 T cells in secondary lym- rophages, proliferation of intrinsic glomerular cells, accumula- phoid tissues. However, the functional contribution of tion of cells and fibrin in Bowman’s space (“crescents”), and MHC II on resident cells to the effector phase of cell- rapid deterioration of renal function. Studies of crescentic mediated immune injury targeted at specific tissues in vivo forms of human (6, 7) and experimental GN (8, 9) suggest an is unknown. MHC II expression can be stimulated on cells important effector role for CD41 T cells. Glomerular deposi- tion of Ig is often sparse or absent in crescentic human GN (7), and in an experimental model, crescent formation has S. Li and C. Kurts contributed equally to this paper. been demonstrated to be antibody independent (10). How- 597 J. Exp. Med. The Rockefeller University Press • 0022-1007/98/08/597/06 $2.00 Volume 188, Number 3, August 3, 1998 597–602 http://www.jem.org Published August 3, 1998 ever, the role of glomerular MHC II expression in the local two-layer peroxidase technique as described (10). The primary immune effector response is unknown. antibodies were H129 (monoclonal anti–mouse CD4; American MHC II-deficient mice have near complete elimination Type Culture Collection, Rockville, MD) and M1/70 (mono- of mature CD41 T cells in peripheral lymphoid organs and clonal anti–mouse Mac-1; American Type Culture Collection). do not develop MHC II–dependent immune reactions Sections of spleen provided a positive control, and protein G–puri- fied rat Ig was substituted for primary mAb to provide a negative (11). B cell responses to T cell–independent but not T cell– control. A minimum of 20 equatorially sectioned glomeruli were dependent antigens are preserved. These mice were used to assessed per animal, and the results were expressed as cells per determine the requirement for MHC II for development of glomerular cross section (c/gcs). crescentic GN in a CD41 T cell–dependent murine model, Assessment of the Systemic Immune Response to Sheep Globulin. initiated by a nephritogenic antigen planted on the glomer- Cutaneous delayed-type hypersensitivity (DTH) to the sheep ular basement membrane (GBM). The contribution of globulin was assessed by intradermal challenge with 0.5 mg in 20 MHC II on intrinsic renal cells was studied in chimeric ml of PBS into the hind foot of mice, 20 d after the systemic ad- mice, which had functional CD41 T cells but did not ex- ministration of sheep anti–mouse GBM globulin, as described press MHC II on intrinsic renal cells. previously (14). Ovalbumin (Sigma-Aldrich Pty. Ltd., Castle Hill, NSW, Australia) injected into the opposite foot pad was used as a control. Circulating titers of mouse anti–sheep globulin antibody were measured by ELISA as described previously (10, 14). Opti- Materials and Methods cal densities (405 nm) were converted to micrograms per millili- Mice. MHC II–deficient (MHCII2/2) mice, generated by tar- ter of antibody by reference to a standard curve generated with geted disruption of the MHCII Aa gene in C57BL/6 mice as de- known concentrations of bound mouse IgG. Serum from six scribed previously (11), were housed under specific pathogen– nonimmunized mice was used as normal control. Isotypes of free conditions. anti–sheep globulin antibody were also measured by ELISA at a Generation of MHC II Chimeric Mice. Chimeric mice (B6→ single dilution of mouse serum optimized for each isotype (1 in MHC2/2) expressing MHC II on bone marrow–derived cells but 20 for IgG2a, 1 in 40 for IgG1, IgG2b, and IgG3, and 1 in 80 for Downloaded from not in other organs were generated using previously described bone IgM) using horseradish peroxidase–conjugated goat anti–mouse marrow and thymus transplantation protocols (12). MHCII2/2 mice IgG1, IgG2a, IgG2b, IgG3, and IgM (Southern Biotechnology were thymectomized at 4 wk of age, irradiated with 700 rad at 7 Associates, Inc., Birmingham, AL) at a dilution of 1:4,000. wk of age, and transplanted with T cell–depleted bone marrow Experimental Design and Statistical Analysis. The development from B6 (C57BL/6) mice. After an additional 4 wk, mice were of GN 21 d after anti-GBM globulin administration was studied engrafted under the kidney capsule with a thymus from an irradi- in the following groups of mice: B6 (C57BL/6) mice (n 5 7), MHCII2/2 mice (n 5 8), B6→MHCII2/2 chimeric mice ated (900 rad) newborn wild-type (WT) mouse. B6 mice were on April 9, 2017 subjected to an identical transplantation process to generate con- (MHCII1/1 thymus and bone marrow in MHC2/2 mice; n 5 8), trol (B6→B6) chimeras. and B6→B6 (control) chimeric mice (MHCII1/1 thymus and Assessment of Lymphocyte Populations in Chimeric Mice. Lympho- bone marrow in MHCII1/1 mice; n 5 8). In addition, measure- cyte subsets in pooled spleen and LN cell suspensions were as- ments of baseline renal function were performed on the following sessed by flow cytometry using the following mAbs: FITC-con- separate groups of nonnephritic mice: B6 (C57BL/6) mice (n 5 jugated anti-B220, PE-conjugated anti-CD4, PE-conjugated 8), MHCII2/2 mice (n 5 7), and B6→B6 chimeric mice (n 5 8). anti-CD8, and FITC-conjugated anti-CD3 (PharMingen, San Results are presented as mean 6 SEM. Statistical analysis was per- Diego, CA) as described previously (10). MHC II expression on formed by one-way analysis of variance, with post hoc analysis B cells was assessed using FITC-conjugated anti-MHCII (Phar- using Fisher’s protected least significant differences (PLSD) test. Mingen). Induction of anti-GBM GN and Assessment of Renal Injury. Crescentic GN was initiated in mice by a single intravenous in- Results jection of 10 mg of sheep anti-GBM globulin, as described previ- Development of Crescentic GN is MHC II Dependent. B6 ously (13). Renal injury and other parameters were assessed 21 d (MHCII1/1) mice developed severe proliferative GN (Fig. later. Histological assessment was performed on 2-mm periodic 1 A) with crescents in 56.5 6 5.2% of glomeruli. This re- acid-Schiff–stained kidney sections. Crescent formation was as- sulted in significant proteinuria (15.5 6 1.5 mg/24 h; base- sessed in a minimum of 30 glomeruli, in a blinded protocol, as described previously (13, 14). Proteinuria was determined by the Bradford method over the final 24 h of each experiment as de- scribed (15). Creatinine clearance was calculated from the serum Table 1.