Background

Increased numbers of lymphocytes have been identified in biopsy specimens of human mesangial proliferative glomerulonephritis (GN). However, the causal relationship between infiltrating T lymphocytes and mesangial changes in mesangial proliferative GN has not been previously evaluated. In this study, we elucidated the role of lymphocytes in the development of mesangial proliferative GN.

Method

Immunohistological and flow cytometric analyses as well as a reverse transcription-polymerase chain reaction (RT-PCR) studies were performed in monoclonal antibody (mAb) 1-22-3–induced Thy 1.1 GN. To elucidate the role of these lymphocytes, depletion studies were carried out using anti-CD8 mAb (OX-8), which depletes both CD8⁺ T lymphocytes and natural killer (NK) cells and anti-CD5 mAb (OX-19), which depletes both CD4⁺ and CD8⁺ T lymphocytes.

Results

Immunofluorescence (IF) studies revealed that NK cells and CD4⁺ T lymphocytes were recruited into glomeruli. Glomerular mRNA expression for interferon-, interleukin-2 (IL-2), IL-10, and perforin increased after induction of GN. Increased expressions of several chemokines, which have the potential to attract lymphocytes, were also detected. Anti-CD8 mAb treatment completely prevented the recruitment of NK cells; however, it had no protective effect on proteinuria and mesangial injury. By contrast, anti-CD5 mAb treatment suppressed the recruitment of CD4⁺ T lymphocytes into glomeruli and reduced proteinuria (60.4 25.7 vs. 120.0 32.3 mg/day, P < 0.05) and mesangial changes evaluated by total number of cells in glomeruli (63.2 6.0 vs. 81.4 5.9, P < 0.01) and -smooth muscle actin staining score (1.4 0.2 vs. 2.2 0.4, cf2P < 0.01) on day 14 after induction of GN. mRNA expression for IL-2 was significantly reduced by OX-19 treatment.

Conclusion

T lymphocytes participate in the development of mesangial proliferative GN.

METHODS

Animals

All experiments were performed using female Wistar rats weighing 150 to 200 g and were purchased from Charles River Japan (Atsugi, Japan). All animal experiments conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Experimental protocol

Experiment 1: Examination of glomerular infiltration by lymphocytes

Thy 1.1 GN was induced in 30 rats by a single injection with 1.0 mL saline containing 500 g of mAb 1-22-3 intravenously through the tail. Preparation of mAb 1-22-3 and induction of Thy1.1 GN have been described previously^17,18,19,20. The rats were sacrificed just before injection of mAb 1-22-3 and at 30 minutes and on days 1, 3, 7, and 14 after induction of GN (N = 5 per time point). At each time point, the right kidney was removed, weighed, and used to prepare the total glomerular RNA. The left kidney was perfused via the aorta with phosphate-buffered saline before being removed and then cut into portions and used for assessment by light microscopy (LM) or IF. The presence of lymphocytes in glomeruli was determined by IF staining using specific mAbs against rat lymphocyte antigens.

Flow cytometric analysis was carried out to analyze the time course of the number of circulating lymphocyte subsets. Ten rats were divided into two groups, and in each group, five rats each were injected with mAb 1-22-3 or saline. To prepare peripheral blood mononuclear cells (PBMCs), heparinized blood was obtained from each rat before injection, and on days 1, 3, 7, and 14 after injection of mAb 1-22-3.

To determine the nature of the cytokines considered to be produced by lymphocytes [interferon- (IFN-), interleukin (IL)-2, IL-4, IL-10, and perforin] and chemokines, which are considered to attract lymphocytes [monocyte chemoattractant protein-1 (MCP-1), regulated on activation, normal T cell expressed and secreted (RANTES), and lymphotactin]^21,22,23, RT-PCR studies were performed on glomerular RNA. The total glomeruli were isolated from pooled kidneys of five rats by a sieving method²⁴.

Experiment 2: Lymphocyte depletion study

In vivo T-cell depletion was induced by injections of the following antilymphocyte antibodies: OX-19, which depletes CD5⁺ pan T lymphocytes^8,25,26, OX-38, which depletes CD4⁺ T lymphocytes^10,27,28, and OX-8, which depletes both CD8⁺ T lymphocytes and NK cells^8,10. As a control, murine IgG1 mAb, RVG1 (against rotavirus) was used²⁹. The rats were injected with mAbs intraperitoneally (3 mg/100 g body weight) and intravenously (1 mg/100 g body weight) simultaneously two days before the induction of GN.

A total of 60 rats was used in this experiment. For OX-19–depletion study, 20 rats were divided into an OX-19–treated group and a control group. Rats were sacrificed on days 7 (N = 5) and 14 (N = 5), and kidneys were removed in the same manner as in experiment 1. Twenty-four–hour urine samples were collected on days 1, 3, 5, 7, 10, and 14 after injection of mAb 1-22-3. Urine protein concentrations were determined by colorimetric assay (BioRad, Oakland, CA, USA) using bovine serum albumin as a standard. OX-38 depletion and OX-8 depletion studies were also designed as an OX-19-depletion experiment. Glomerular injury was assessed by LM, IF, and kidney weight. The number of glomerular infiltrating lymphocytes on day 7 was counted to assess the effects of OX-8 treatment on the recruitment of lymphocytes in glomeruli.

Flow cytometric analysis was carried out to assess the effects of mAb treatments on circulating lymphocytes. Five rats were used for each depletion group. To prepare PBMCs, heparinized blood was obtained from each rat on days 9 and 16 after antilymphocyte mAb treatments.

To further analyze the role of CD5⁺ lymphocytes, 60 rats were divided into an anti-CD5 mAb (OX-19)–treated group and a control group treated with RVG1, a murine mAb against rotavirus. The rats from each group were sacrificed before injection and at 30 minutes and on days 1, 3, 7, and 14 after an injection of mAb 1-22-3 (N = 5 on each time point), and kidneys were removed as described in experiment 1. The fixation of complements in glomeruli was compared as described before¹⁹. The decreased level of serum CH50 in each group was also compared 30 minutes after injection of mAb 1-22-3. The time courses of glomerular infiltration of lymphocytes and monocytes/macrophages were compared by IF staining, with specific mAbs against rat lymphocytes antigen and mAbs against macrophage subsets. At each time point, expression of mRNA for lymphokines (IFN-, IL-2, IL-10, and perforin), platelet-derived growth factor (PDGF)-BB, and transforming growth factor- (TGF-) was also compared by RT-PCR on isolated glomeruli of pooled kidneys from five rats.

Another set of experiments was carried out to quantitate the glomerular mRNA for IL-2 and IFN-. Ten rats were divided into an anti-CD5 mAb-treated group and a control group. Five rats of each group were sacrificed on day 14 after injection of mAb 1-22-3. Glomerular RNA was prepared from each animal and was used for the RT-PCR study.

To compare the amounts of mAb 1-22-3 bound to the kidney, three rats from each group were injected with mAb 1-22-3 labeled with ¹²⁵I by the chlolamine-T method³⁰. The radioactivity was measured with an autogamma scintillation photometer (model 5260; Packard, Sterling, VA, USA) 30 minutes after injection.

Light microscopy

Tissue samples for light microscopic assessment were fixed with 10% neutral-buffered formalin, embedded in paraffin, cut into 4 m sections, and stained with periodic acid-Schiff (PAS) reagent. Semiquantitative morphological studies of glomerular lesions were carried out by randomly selecting 30 full-sized glomeruli (80 to 100 m) from each specimen. The sections were analyzed in a double-blind manner, and the degree of glomerular mesangial matrix expansion was scored 0 to 4+ according to the percentage of glomerular involvement, as described by Raij, Azar, and Keane³¹. The total number of cells in glomeruli was also counted in a blind protocol and computed for 30 glomeruli for each kidney.

Immunofluorescence

Tissue samples for IF studies were snap frozen in precooled n-hexane and stored at -70°C. Frozen sections 3 m thick were cut with a cryostat and stained with specific mAbs to rat lymphocytes and monocytes/macrophages. Mouse anti-rat mAb OX-19 (IgG1, anti-CD5)^8,25,26 was used as pan T-cell marker, OX-38 (IgG2a, anti-CD4)^10,27,28 as a T helper cell marker, OX-8 (IgG1, anti-CD8)^8,10 as an T cytotoxic/suppressor cell marker, and 10/78 (IgG1, anti-NKR-P1) as an NK cell marker. OX-19, OX-38, and OX-8 were precipitated from ascites using the corresponding hybridoma (European Collection of Animal Cells, Porton Down, Salisbury, UK); 10/78 was purchased from Serotec (Oxford, UK). ED1 (IgG1, reactive with pan monocytes/macrophages) and ED3 (IgG2a, reactive with macrophage sialoadhesin) were used to detect monocytes and macrophages purchased from Chemicon International Inc. (Temecula, CA, USA) and Serotec, respectively. Fluorescein isothiocyanate (FITC)-conjugated goat antimouse IgG1 and FITC-conjugated goat anti-mouse IgG2a were used as secondary antibodies (Southern Biotechnology Associates, Birmingham, AL, USA). The number of mononuclear cells per glomerular cross-section (c/gcs) was counted in 50 randomly selected full-sized glomeruli by an observer who was unaware of the experimental protocol. Double-staining IF studies with OX-19 and OX-38, with OX-19 and R1-10B5 (IgG2a, anti-CD8; Seikagaku Co., Tokyo, Japan), or with 10/78 and R1-10B5 were performed. Tetramethyl-rhodamine isothiocyanate (TRITC)-conjugated goat anti-mouse IgG1 (for OX-19 and 10/78) and FITC-conjugated anti-mouse IgG2a (for OX-38 and R1-10B5) were used as secondary antibodies (Southern Biotechnology Associates). Double staining with anti-rat perforin (Torrey Pines Biolabs, Inc., San Diego, CA, USA), and 10/78 was performed using FITC-conjugated anti-rabbit IgG (Dako, Glostrup, Denmark) and TRITC-conjugated goat anti-mouse IgG1 as secondary antibodies. To quantitate the mesangial changes, frozen sections were stained with anti-collagen type I (Chemicon) or -smooth muscle actin (-SMA; IgG2a; Sigma, St. Louis, MO, USA) antibodies. The degree of collagen type I and -SMA staining was scored 0 to 4+ in 30 randomly selected glomeruli according to the method described by Floege et al³². FITC-conjugated anti-rabbit IgG was used as secondary antibody to detect rabbit anti-rat collagen type I. FITC-conjugated anti-rat C3 (Cappel, West Chester, PA, USA) was used to assess the glomerular fixation of complement.

Flow cytometric analysis

The relative lymphocyte subset frequencies in the peripheral blood of rats were analyzed by flow cytometry. To prepare PBMCs, heparinized artery blood was obtained from tail arteries when the animals were under general anesthesia. The total mononuclear cells in each blood sample were counted in triplicate with a hemacytometer after being diluted with Turk's solution. The contaminating erythrocytes in the cell preparation were lyzed by 0.85% ammonium chloride. FITC-conjugated OX-19, OX-38, and OX-8 prepared as described by Kawamura³³, and FITC-conjugated 10/78 (Serotec) were used to detect lymphocyte subsets. Cytometric analysis of surface antigen expression was performed on a FACScan (Becton Dickinson, Mountain View, CA, USA). Total leukocytes were detected using the OX-30 mAb (antileukocyte common antigen marker; Caltag, Burlingame, CA, USA), and RVG1 (mouse IgG1) was used as a negative control.

RT-PCR

Isolated glomeruli were immediately dissociated by guanidinium and phenol extraction (TRIZOL; GIBCO BRL, Gaithersburg, MD, USA). Complementary DNA (cDNA) was synthesized using a commercial kit (SuperScript Preamplification System; GIBCO BRL) following the standard protocol. The primers were designed according to published sequences Table 1. Amplification was carried out using the PC-800 programmable temperature control system (Astec, Fukuoka, Japan) through 20 to 40 cycles of denaturation at 95°C for 30 seconds, annealing at individual temperatures for 30 seconds, and extension at 72°C for 10 minutes. The optimal cycle numbers were determined in a preliminary trial to be in the linear phase of amplification. Amplification of the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was used as a positive control for intact RNA and for measuring the efficiency of RT. Negative controls without cDNA and positive controls of cDNA from Con-A–stimulated rat spleen cells were included in all experiments. The PCR products were subjected to electrophoresis through 1.5% agarose gels and were stained with ethidium bromide. The band intensities were determined by image analysis using a Macintosh computer and densitometry software (Densitograph; ATTO, Tokyo, Japan).

Table 1 - Polymerase chain reaction (PCR) primers used in this study.

Full table

Statistical analysis

Statistical significance was evaluated using the unpaired t-test or the Mann–Whitney U-test. Values were expressed as the mean SD. Differences at P < 0.05 were considered significant. Data were analyzed using StatView for Macintosh (Abacus Concepts, Berkeley, CA, USA).

RESULTS

Experiment 1: Glomerular infiltration of lymphocytes

Significant increases in levels of lymphocytes, indicated by the presence of OX-19 (CD5)⁺, OX-38 (CD4)⁺, OX-8 (CD8)⁺, and 10/78 (NKR-P1)⁺ cells were observed in glomeruli and peaked on day 7 after injection of mAb 1-22-3 Figure 1. In the double-staining study, OX-38⁺ cell were also stained with OX-19 Figure 2a. On the other hand, almost all CD8⁺ cells in glomerulus were also stained with 10/78 Figure 2c and were negative for OX-19 Figure 2b. At this time point, part of NKR-P1⁺ cells were stained by the antiperforin mAb Figure 2d. Significant decreases in total numbers of mononuclear cells, total T cells (CD5⁺ cells), CD4⁺ cells, CD8⁺ cells, and NKR-P1⁺ cells after an injection of mAb 1-22-3 were revealed by flow cytometric analysis Figure 3. RT-PCR revealed increased expression of mRNA for lymphokines, IFN-, IL-2, IL-10, and perforin, but not for IL-4 after an injection of mAb 1-22-3 Figure 4. Expression of the chemokines MCP-1, RANTES, and lymphotactin also increased Figure 4.

Figure 1.

Time course of glomerular infiltration of lymphocytes after an injection of monoclonal antibody (mAb) 1-22-3. Numbers of lymphocytes per glomerular cross section were counted in 50 randomly selected full-size glomeruli, and the results were expressed as mean SD (N = 5). Symbols are: () OX-19(CD5)⁺ cells; () OX-38 (CD4)⁺ cells; () OX-8 (CD8)⁺ cells; () 10/78 (NKR-P1)⁺ cells; *P < 0.05 and **P < 0.01 compared with before injection.

Full figure and legend (16K)

Figure 2.

Double staining immunofluorescence (IF) studies of rat glomeruli seven days after the induction of glomerulonephritis (GN). (A) Double staining with anti-CD5 mAb (OX-19) and anti-CD4 mAb (OX-38). All CD5⁺ cells were also stained with anti-CD4 mAb (yellow). (B) Double staining with OX-19 (red) and anti-CD8 mAb (R1-10B5; green). No double positive cells were detected. (C) Double staining with R1-10B5 and anti-NKR-P1 mAb (10/78). All CD8⁺ cells were also stained with anti-NKR-P1 mAb (yellow). (D) Double staining with antirat perforin and 10/78. Part of NKR-P1⁺ cells (red) were also stained with antiperforin (arrowhead, yellow). Original magnification, 400.

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Figure 3.

Kinetics of the number of circulating lymphocytes after injection of rats with mAb 1-22-3. Symbols are: () saline group; () mAb 1-22-3 group. Absolute subset numbers were calculated using leukocyte counts, and the proportions of subsets were determined by flow cytometry. Significant reductions of circulating total mononuclear cells (A), CD5 (OX-19)⁺ cells (B), CD4 (OX-38)⁺ cells (C), CD8 (OX-8)⁺ cells (D), and NKR-P1(10/78)⁺ cells (E) were observed by day 14 after injection of mAb 1-22-3. Values are expressed as mean SD (N = 5). *P < 0.05 and **P < 0.01 compared with saline-injected controls.

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Figure 4.

Expression of rat glomerular mRNA for lymphokines and chemokines after injection with mAb 1-22-3. The data shown are representative semiquantitative measurements made by RT-PCR amplification of mRNA from five rats. mRNA from five rats sacrificed at each time point was pooled and used for RT-PCR. The representative agarose gel electrophoretic patterns from one of three independent experiments are shown at top. Ratios of the densitometric signals of cytokines to that of the internal control (GAPDH) were computed. A weak signal was detected in normal control rats for all cytokines studied. The data are shown as ratios relative to normal rat findings and are expressed as mean SD of three independent experiments. Increased expression for lymphokines (A, B, D, and E), except for IL-4 (C), and those for chemokines (F–H) was detected after induction of Thy 1.1 GN.

Full figure and legend (100K)

Experiment 2: Lymphocyte depletion study

Cell numbers in circulating lymphocyte subpopulations in leukocyte-depleted and control groups were summarized in Table 2. In peripheral blood, anti-CD5 mAb (OX-19) treatment caused marked reductions in the numbers of CD5⁺ and CD4⁺ cells, which were maintained until the end of the experiment. Treatment with anti-CD4 mAb (OX-38) also reduced the number of CD4⁺ cells; however, the effect was mild compared with that of anti-CD5 mAb treatment. Anti-CD8 mAb (OX-8) affects the numbers of both CD8⁺ cells and NKR-P1⁺ cells but had no effect on CD4⁺ cell count. Circulating lymphocyte populations in control (RVG1)-treated rats were unaffected.

Table 2 - Cell numbers in circulating lymphocyte subpopulations in lymphocyte-depleted and control-treated rats.

Full table

The change in degree of proteinuria over time is illustrated in Figure 5. Significant reduction of proteinuria on days 5, 7, and 10 was observed in the anti-CD5 mAb (OX-19) and anti-CD4 mAb (OX-38)–treated groups. A comparison of mesangial injury evaluated by total number of cells in glomeruli, matrix score, collagen type I staining score, -SMA staining score, and kidney weight on day 14 is summarized in Figure 6. Reduced mesangial injury was observed only in the anti-CD5 mAb (OX-19)–treated group Figure 6 and Figure 7. Although anti-CD8 mAb treatment reduced the recruitment of CD8⁺ cells (0.061 0.017 vs. 4.58 0.82, P < 0.01) and NKR-P1⁺ cells (0.11 0.062 vs. 4.26 1.06, P < 0.01) seven days after induction of GN, no significant changes was observed in the anti-CD8 mAb-treated group compared with the control group.

Figure 5.

Effects of anti-CD5 mAb (OX-19; A and B), anti-CD4 mAb (OX-38; C and D) and anti-CD8 mAb (OX-8; E and F) treatment on kinetics of proteinuria in rats after injection of mAb 1-22-3. A significant reduction of proteinuria was observed in the anti-CD5 mAb– and anti-CD4 mAb–treated group. Each study was carried out twice and followed for 7 (B, D, and F) or 14 (A, C, and E) days after injection of mAb 1-22-3. Symbols are: () control group; () anti-CD5 mAb (OX-19)-treated group; () anti-CD4 mAb (OX-38)-treated group; () anti-CD8 mAb (OX-8)-treated group. The results are expressed as mean SD (N = 5). *P < 0.05 and **P < 0.01 compared with the control group at the same time point.

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Figure 6.

Effect of lymphocyte depletion on rat glomerular changes. Glomerular changes evaluated by total number of cells per glomerular cross section (c/gcs) (A), matrix expansion (B), collagen type I staining score (C), -smooth muscle actin (-SMA) staining score (D), and kidney weight (E) were compared between the lymphocyte-depleted group and the control group on day 14. Each value is expressed as mean SD (N = 5). Symbols are: () control group; () anti-CD5 mAb treated group; () anti-CD4 mAb treated group; () anti-CD8 mAb treated group; *P < 0.05; **P < 0.01.

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Figure 7.

Micrographs of periodic acid-Schiff (PAS)-stained kidney sections from rats treated with anti-CD5 mAb (OX-19; A) and from a control (RVG1-treated) group (B) 14 days after injection of mAb 1-22-3. Original magnification, 200.

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Further analysis of the effects of anti-CD5 mAb (OX-19) treatment

To ascertain whether the anti-CD5 mAb treatment might interfere with the binding of mAb 1-22-3 to kidney, the radioactivity of ¹²⁵I-labeled mAb 1-22-3 was measured in a -counter. In the group of rats treated with anti-CD5 mAb, the mean amount of mAb 1-22-3 bound in a kidney was 7.84 0.54 g, which was comparable to the 7.92 0.079 g bound to kidney in the RVG1-treated control group, and there were no significant differences between these two groups (P = 0.51). To exclude the possibility that anti-CD5 mAb treatment interferes with the fixation of complement in glomeruli, we used the autoexposure system to measure fluorescence intensities of FITC-conjugated antirat C3 in glomeruli from rats 30 minutes after an injection of mAb 1-22-3. There were no differences in exposure time for the fluorescence intensities of FITC-conjugated C3 deposition in the glomeruli (9.37 1.25 vs. 9.52 1.24 s, P = 0.85). The serum CH50 values just before an injection of mAb 1-22-3 (50.23 1.48 vs. 49.18 2.06 U/mL, P = 0.44) and after an injection of mAb 1-22-3 (35.58 2.69 vs. 33.58 1.18 U/mL, P = 0.22) also showed no significant difference between the initiation of anti-CD5 mAb treatment and control group.

The IF study revealed that treatment of rats with anti-CD5 mAb affected glomerular accumulation of CD4⁺ cells, whereas this treatment did not influence the number of NKR-P1⁺ cells. In addition, anti-CD5 mAb treatment did not influence the number of ED1⁺ cells (pan monocytes/macrophages), but a significant reduction of ED3⁺ cells (activated macrophages) was observed Figure 8.

Figure 8.

Effects of anti-CD5 mAb (OX-19) treatment on rat glomerular leukocyte accumulation after an injection of mAb 1-22-3. Time course of the extent of glomerular infiltration by OX-19 (CD5)⁺, OX-38 (CD4)⁺, OX-8 (CD8)⁺, and 10/78 (NKR-P1)⁺, ED1⁺, and ED3⁺ cells was compared between the anti-CD5 mAb-treated group () and the RVG1-treated control group () by IF staining. Values are expressed as mean SD. *P < 0.05; **P < 0.01 compared with the control group at the same time point.

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To examine the cytokine profile, mRNA from isolated glomeruli was compared with that from the control group Figure 9. GAPDH RT-PCR data confirmed that similar concentrations of cDNA were analyzed from each sample. Anti-CD5 mAb treatment suppressed expression of glomerular IL-2 mRNA through the experimental period. mRNA expression for IFN- was reduced on day 14. This treatment did not reduce the glomerular expression of IL-10 or perforin mRNA. Glomerular expression of PDGF or TGF- mRNA increased immediately after an injection of mAb 1-22-3; however, anti-CD5 mAb had no effect on glomerular expression of these cytokines.

Figure 9.

Effect of anti-CD5 mAb (OX-19) treatment on rat glomerular mRNA expression for cytokines after Thy1.1 GN induction. mRNA expression for cytokines was measured by RT-PCR on total RNA pooled from five rats from anti-CD5 mAb-treated group (GN) and RVG1-treated control group (). The ratios of the densitometric signals of cytokine mRNA relative to the internal control (GAPDH) were computed. The data are shown as ratios relative to normal rat findings and expressed as mean SD.

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Reverse transcription-PCR analysis using glomerular RNA from individual rat also demonstrated that both IL-2 (0.80 0.29 vs. 2.03 1.55, P < 0.01) and IFN- mRNA expression (0.42 0.13 vs. 0.64 0.15, P < 0.05) were reduced in the anti-CD5 mAb-treated group on day 14.

DISCUSSION

Previously, we reported that increased numbers of lymphocytes were observed in the mesangial area in Thy 1.1 GN on electron microscopy²⁰. These lymphocytes in the mesangial area seem to be involved in mesangial cell proliferation and the consequent alterations to the mesangium. In this study, we elucidated the role of various lymphocytes in the development of Thy 1.1 GN. IF studies with mAbs that recognize subpopulations of lymphocytes revealed that numerous CD8 (OX-8) and NKR-P1 (10/78)-positive cells were recruited into glomeruli beginning just after anti-Thy 1.1 mAb injection. Infiltration of T lymphocytes recognized by anti-CD5 mAb (OX-19) or anti-CD4 mAb (OX-38) was also detected, which were fewer than CD8⁺ or NKR-P1⁺ cells. OX-8 is reported to recognize both CD8⁺ T lymphocytes and NK cells in rats^8,10,38,39. Double-staining IF study revealed that most OX-8⁺ cells detected in glomeruli were NK cells rather than CD8⁺ T lymphocytes Figure 2c and that almost all T lymphocytes (CD5⁺ cells) infiltrated in glomeruli were CD4⁺ T lymphocytes Figure 2a. From these findings, we concluded that mainly NK cells and CD4⁺ T lymphocytes infiltrate glomeruli in Thy 1.1 GN. Flow cytometric analysis showed that the number of circulating lymphocytes decreased after an injection of mAb 1-22-3. Our previous study revealed that there were no inflammatory lesions in any other organs to which mAb 1-22-3 binds, such as thymus, lymph nodes, spleen, liver, or intestines. These observations indicate that circulating lymphocytes are recruited into the inflammatory site of the kidney after an injection of mAb 1-22-3. RT-PCR study demonstrated that the expression of glomerular mRNA for certain cytokines that are considered to be produced by lymphocytes, such as IFN-, IL-2, IL-10, and perforin, increased after mAb 1-22-3 injection. These findings also supported that idea that lymphocytes are recruited into glomeruli.

Thy 1.1 GN is induced by passive injection of anti-Thy 1.1. mAb without immunization with specific antigen. The mechanism of the recruitment of T lymphocytes and NK cells into glomeruli is unclear in this GN model. It was reported that certain chemokines could attract T lymphocytes to the inflammatory site without specific antigen presentation^40,41. Several studies showed that MCP-1 and RANTES could attract CD4⁺ and CD8⁺ T lymphocytes, while lymphotactin is considered to be the chemoattractant specific for CD8⁺ T lymphocytes and NK cells^21,22,23. In this study, we revealed that expression of glomerular mRNA for the chemokines MCP-1, RANTES, and lymphotactin increases. Recently, it was reported that both MCP-1 and RANTES can activate T lymphocytes without involving T-cell antigen receptor signaling^41,42,43. Stahl et al reported that MCP-1 was produced by mesangial cells that were stimulated by anti-Thy 1.1 antibody binding⁴⁴. Thus, it is conceivable that non–antigen-specific T lymphocytes are recruited by the chemokines from glomerular cells.

In this study, we demonstrated that the numerous NK cells were recruited in glomeruli. A double-staining IF study revealed that most NK cells in glomeruli produced perforin, a major participant in the lytic machinery of cytotoxic lymphocytes⁴⁵. However, anti-CD8 mAb (OX-8) treatment had no protective effect on proteinuria Figure 5 or any morphological parameters Figure 6. IF findings showed that anti-CD8 mAb treatment completely prevented the recruitment of NK cells into glomeruli. These findings indicating that NK cells did not play an active role in the pathogenesis of Thy 1.1 GN. On the other hand, anti-CD5 mAb (OX-19) treatment clearly reduced proteinuria and reduced the mesangial injury, as evaluated by total number of cells in glomeruli, matrix score, -SMA staining score, and collagen type I staining, as well as by increased kidney weight on day 14 after induction of Thy 1.1 GN Figure 5 and Figure 6. We believe that these findings indicate that anti-CD5 mAb treatment suppresses mesangial cell proliferation and consequent mesangial matrix expansion. Anti-CD4 mAb treatment also reduced proteinuria but did not suppress mesangial injury. Because double-staining IF findings indicated that most T lymphocytes in glomeruli are of the CD4⁺ subtype Figure 2a, it is unclear why only anti-CD5 mAb (OX-19) but not anti-CD4 mAb (OX-38) treatment prevents mesangial injury. It might be explained by the difference in efficiency of depletion of CD4⁺ T lymphocytes by OX-19 and OX-38. Flow cytometric analysis showed that anti-CD5 mAb treatment reduced the number of circulating CD4⁺ T lymphocytes to around 20% and that this suppression was maintained throughout the experiment Table 2. However, CD4⁺ T lymphocyte depletion by anti-CD4 mAb treatment was not as effective, and CD4⁺ T-lymphocyte levels recovered earlier after this treatment than after anti-CD5 mAb treatment Table 2. Some other reports have also mentioned difficulty in depleting CD4⁺ T lymphocytes with anti-rat CD4-specific mAbs^28,46. Thus, it is considered that the effect of treatment with OX-38 might not be sufficient to reduce the mesangial injury as evaluated on day 14. However, we cannot completely rule out the possibility that OX-19⁺/OX-38^- cells contribute to the development of mesangial injury.

The mechanism of the protective effect of anti-CD5 mAb (OX-19) in the development of Thy 1.1 GN is not known. However, some findings obtained in this study might explain this observation. We showed here that anti-CD5 mAb treatment did not influence ED1⁺ cell recruitment into glomeruli, while it suppressed ED3⁺ cell recruitment Figure 8. ED3 is known to detect macrophage sialoadhesin, a marker of activated macrophages, which may participate in the pathogenesis of inflammatory disease, including GN^47,48,49. We previously reported that TRPM-3⁺ macrophages accumulated in glomeruli in Thy 1.1. GN⁵⁰. It is reported that TRPM-3 has the same specificity for the macrophage subpopulation as does ED3⁵¹. In that study, we mentioned that TRPM-3⁺ cells are possibly more intimately related with the progression of Thy 1.1 GN than are ED1⁺ cells. The finding that anti-CD5 mAb treatment decreased the number of ED3⁺ cells in glomeruli indicated that CD5⁺ lymphocytes contribute to the attraction and/or activation of ED3⁺ cells. Thus, one explanation for the mechanism of protective effect of anti-CD5 mAb is that it reduces mesangial injury by down-regulating the function of macrophages.

Although we have demonstrated in this study that anti-CD5 mAb treatment decreased some morphological parameters to 70 to 80%, this treatment could not normalize them. This suggests that not only T-lymphocyte–associated factors but also other factors such as PDGF or TGF-, which were not affected with anti-CD5 mAb treatment Figure 9, play some roles in development of GN. We showed that anti-CD5 mAb treatment reduced the expression of glomerular mRNA for IL-2 and IFN- Figure 9, although the roles of these cytokines in the development of Thy 1.1 GN were not determined in this study. IL-2 and IFN- are known to have multifunctional effects on the inflammatory process^52,53. We believe that investigating the interaction between these cytokines and mesangial cells is important for clarifying the mechanism of mesangial injury. Our depletion study clearly demonstrates that lymphocytes participate in the development of Thy 1.1 GN. However, because we analyzed the role of lymphocytes in the reversible model induced by a single injection of mAb 1-22-3 in this study, the long-term consequences of the anti-CD5 mAb treatment remain to be determined. We previously reported the irreversible progressive models induced by two consecutive injections of mAb 1-22-3⁵⁴ or a single injection to unilaterally nephrectomized rats⁵⁵. The roles of lymphocytes in these progressive models should be investigated in the future studies.

In conclusion, we demonstrate that treatment with anti-CD5 mAb (OX-19) reduces proteinuria and glomerular injury in Thy1.1 GN in rat, which suggests that T lymphocytes contribute to the pathogenesis of human proliferative GN.

References

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Acknowledgments

This work was supported by Grant-Aids for Scientific Research (c) (11671031 to H. Kawachi) and Grant-Aids for Scientific Research (B) (08457286 to F. Shimizu) from the Ministry of Education, Science, Culture, and Sports of Japan. The authors express their gratitude to Dr. Yumi Ito, Dr. Hiroko Koike, and Dr. Akihisa Oyanagi for their helpful discussions. The authors also thank Ms. Y. Kondo and Ms. M. Oba for their technical assistance.