- Email: [email protected]
lpr mice
CELLULAR
IMMUNOLOGY
141,496-507
(1992)
Anti-CD4 Monoclonal Antibody Therapy Suppresses Autoimmune Disease in MRL/Mp-lpr/lpr Mice’ DOUGLASA.JABS,*,~ C.LYNNE BUREK,? QILE Hu,? RUDOLFC.KUPPERS,~ BELLALEE,* ANDROBERTA.PRENDERGAST* *The Wilmer Ophthalmological Institute, The Johns Hopkins University School of Medicine; and tthe Department of Immunology and Infectious Diseases, The Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205 Received December 3, 1991; accepted January 16, I992 MRL/Mp-lpr/lpr (MRL/lpr) mice spontaneously develop systemic autoimmune disease, characterized by vasculitis, lymphadenopathy, glomerulonephritis, and autoantibody formation. The target organ inflammatory lesions are composed largely of CD4+ “helper” T cells, while the massivelyenlarged lymph nodes are composed primarily of CD3+ CD4- CD8- TCR a//3’ “doublenegative” T cells. In this study we investigated the effect of treatment of MRL/lpr mice with antiCD4 monoclonal antibody (mAb); control groups consisted of animals treated with normal saline or rat immunoglobulin (Ig). Anti-CD4 mAb treatment, which was started at 4 weeks and continued through 20 weeks of age, resulted in a dramatic reduction of both the frequency and severity of the autoimmune disease, as demonstrated histologically and serologically. Anti-CD4 mAb therapy markedly reduced the frequency of glomerulonephritis and eliminated vasculitis of the major renal arterial branches. Glomerulonephritis was detected in 9 of 9 saline-treated, 9 of 9 rat Igtreated, but in only 1 of 9 anti-CD4 mAb-treated mice; vasculitis was detected in 6 of 9 salinetreated, 7 of 9 rat Ig-treated, but in none of 9xanti-CD4 mAb-treated mice. The frequency of antinuclear antibodies, titer of anti-dsDNA antibodies, and total Ig levels were all significantly reduced by anti-CD4 mAb therapy. These data support the hypothesis that CD4+ T cells play a central role in the disease process in this autoimmune strain. 0 1992 Academic PXSS. I~C.
INTRODUCTION Several strains of autoimmune mice have been described, including MRL/Mp-lpr/ lpr (MRL/lpr)3, MRL/Mp-+/+ (MRL/+), (NZBxNZW) Fl hybrid (NZB/W), BXSB, and Palmerston-North strains (l-4). MRL/lpr spontaneously develop an autoimmune disease characterized by vasculitis, massive lymphadenopathy, glomerulonephritis, and autoantibody formation. The target organ lesions, including vasculitis, are largely ’ This study was supported in part by Grants EY05912, EY03521, EY01765, and AR31632 from the National Institutes of Health, Bethesda, Maryland, and an unrestricted grant from Research to Prevent Blindness. Dr. Jabs is a Research to Prevent Blindness Olga Keith Wiess Scholar. 2 To whom correspondence should be addressed at The Wilmer Ophthalmological Institute, 550 North Broadway, Suite 700, Baltimore, MD 2 1205. 3 Abbreviations used: MRL/lpr, MRL/Mp-lpr/lpr; MRL/+, MRL/Mp-+/+; NZBJW, (NZB X NZW) Fl hybrid; mAb, monoclonal antibody; SCID, severe combined immunodeficiency; Ig, immunoglobulin; FITC, fluorescein isothiocyanate; ANA, antinuclear antibodies; AP, alkaline phosphatase. 496 000%8749192 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form resewed
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composed of CD4+ T cells (5) while the massively enlarged lymph nodes are composed primarily of Thy 1.2+ CD4- CD8- TCR a/P’ “double-negative” T cells (5-8). A previous report by Wofsy et al. (9) has underscored the importance of T cells in the development of disease in MRL/lpr mice, where the autoimmune disease was suppressed by treatment with the anti-T cell monoclonal antibody (mAb), anti-Thy 1.2, as evidenced by decreased autoantibody production, decreased proteinuria, and improved survival. This result was different from that seen in NZB/W mice, where antiT cell mAb treatment did not alter the disease, or in BXSB mice, where such treatment resulted in fatal anaphylaxis (9). While this study emphasized the important role that T cells play in the disease in MRL/lpr mice, it did not distinguish between an effect on CD4+ T cells or on double-negative T cells. Anti-CD4 mAb has been used to treat NZB/W mice and resulted in suppression of their autoimmune disease (10). The discrepancy between the results of treatment with anti-T cell mAb and anti-CD4 mAb in NZB/W mice remains unexplained. Our previous investigation of the vasculitic lesions in the kidneys of MRL/lpr mice revealed that the infiltrating mononuclear cells were primarily T cells, of which 58% were CD4+ and 12% CD8+, together with 5% B cells and 3% macrophages; a similar immunohistologic profile was found in the lacrimal gland lesions (5). This result suggested to us that the beneficial effect of treatment with anti-T cell mAb on the autoimmune lesions in MRL/lpr mice might be due to an effect upon the CD4’ T cell. In addition, anti-CD4 mAb was recently found to suppress autoantibody production and lymphadenopathy in MRL/lpr mice (11). The present study using anti-CD4 mAb therapy demonstrates marked suppression of the autoimmune disease as determined by reversal of the immunopathologic lesions, as well as the serologic abnormalities normally present in older MRL/lpr mice. MATERIALS
AND METHODS
Monoclonal antibodies. Monoclonal antibodies were prepared using the method of Wofsy et al. (9, 10). Hybridoma cells secreting a mAb to the murine CD4 antigen L3T4 (GK 1.5) were obtained from ATCC (Rockville, MD) (12, 13). One million cells were injected into the peritoneal cavity of severe combined immunodeficiency (SCID) mice obtained from Jackson Laboratories (Bar Harbor, ME). Antibody was harvested from the SCID peritoneal exudate by paracentesis starting 8 days after cell transfer. lmmunoglobulin (lg) was partially purified using ammonium sulfate precipitation followed by solubilization and dialysis against phosphate-buffered saline (PBS). The partially purified mAb was then standardized for protein content using spectrophotometric absorbance at 280 nm. Treatment protocol. MRL/lpr mice were obtained from the Jackson Laboratories and kept under standard conditions in the animal facilities of the Woods Research Building of The Johns Hopkins Hospital. Animals were treated starting at 1 month of age with one initial intravenous and one intraperitoneal injection of 2 mg each followed by weekly intraperitoneal injections of 2 mg of anti-L3T4 mAb. Two control groups were used: one was given injections of normal saline, and a second was given injections of rat lg. The rat lg used in these experiments was produced by subjecting normal rat serum to an ammonium sulfate precipitation and dialysis identical to that used for the preparation of the monoclonal anti-L3T4 mAb. At 5 months of age,
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animals were sacrificed by exsanguination. Sera were collected for the analyses outlined below. Proteinuria was measured using dipstick analysis, which is graded in a semiquantitative scale of 0 to 4f (Albustix; Miles Inc., Sebhart, IN). Proteinuria was classified as normal if it was graded 0 or trace and abnormal if it was l+ or greater. Submandibular, axillary, and inguinal nodes were removed and weighed to give a measurement of the lymphadenopathy. Spleens were removed and weighed separately. Kidneys were removed for histologic processing, fixed in 4% buffered formaldehyde, embedded in paraffin, sectioned at 5 pm, and stained with hematoxylin and eosin. Histopathologic evaluation was performed by a masked reader (R.A.P.) unaware of the treatment for each animal. Lymphocyte subset analysis. Animals were sacrificed at 3 months of age for flow cytometry analysis of lymphocyte subset populations of their spleens. Spleens were removed aseptically; a cell suspension isolated; and 4 X 1O6spleen cells were suspended in 1 ml PBS and incubated for 30 min with the primary antibody. Antibodies used included anti-Thy 1.2 (Becton-Dickinson, Mountain View, CA) for T cells, anti-L3T4 (Becton-Dickinson) for CD4+ T cells, and anti-Lyt 2 (Becton-Dickinson) for CD8+ T cells. Cells were washed, resuspended, and incubated for 30 min with a fluorescein isothiocyanate (FITC)-conjugated goat anti-rat antibody (Organon Tekika, West Chester, PA). The percentage of cells staining positively was enumerated using a Facscan flow cytometer (Becton-Dickinson). Negative controls for each analysis used rat Ig for the primary antibody. Antinuclear antibodies. Antinuclear antibodies (ANA) were measured using indirect immunofluorescence. Sera were screened at a 1: 10 dilution, using 4-pm frozen sections of rat liver as the substrate and a FITC-conjugated goat anti-mouse mAb as the second antibody. ANA were read in a masked fashion and scored on a semiquantitative scale score of 0 to 4+ as previously described (14) where 0 is negative, 1+ is weak, 2+ is medium, 3+ is strong, and 4+ is very strong staining. Measurement of anti-DNA antibodies. Determination of anti-double-stranded DNA (anti-dsDNA) antibodies was performed using the method described by Pisetsky and Peters (15). Briefly, ELISA plates (Corning Lab Sciences Co., New York, NY) were coated with 200 ~1 of salmon testes DNA (Sigma, St. Louis, MO, 5 pg/ml in PBS) in each well and incubated at 37°C for 2-3 hr. After decanting and washing the plates three times with PBS-Tween 20, the remaining sites in each well were blocked by incubation with 200 ~1 of 1 mg/ml methylated bovine serum albumin at 25°C for 1 hr. Plates were washed three times with PBS-Tween 20; then serum diluted 1:2 with PBS-Tween 20 was added and the plates were incubated at 25°C for 1 hr. At the end of the incubation, plates were washed three times and 100 ~1 of 1: 1000 dilution of alkaline phosphatase (AP)-coupled goat anti-mouse Ig (whole molecule) or, for subclass determination, goat anti-mouse IgM, IgG, or IgA AP-conjugate (Sigma) was added to each well. After 1-hr incubation at 25°C each well was again washed three times, and then p-phenol phosphate was added as a substrate. Reactions were stopped with the addition of 50 ~1 of 3 M NaOH in each well for 10 min. Each serum was also tested on plates without coating with DNA. Sera from BALB/c and 4-week-old MRL/lpr mice were used as negative controls, while sera from untreated 5-month-old MRL/ lpr mice were used as positive controls. Plates were read using optical density at 410 nm. Autoantibody determinations were performed in a masked fashion.
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Determination of total immunoglobulin. ELISA plates were coated with 100 ~1 of 5 pg/ml goat anti-mouse Ig (IgM, IgG, IgA; Capell, Cochranville, PA) in each well at 4°C overnight. Plates were blocked with 1% BSA; after washing the plates three times with PBS-Tween 20, 100 ~1 of serum at dilutions of 1:2 to 1: 100,000 were added to the individual wells and incubated at 25°C for 1 hr. Relative antibody subclass concentrations were determined using AP-conjugated goat anti-mouse IgG, IgM, or IgA as described above. RESULTS Lymphoprolijkation. Two animals from each group (saline, rat Ig, anti-CD4 mAb treatment) were sacrificed at 3 months of age for analysis of the lymphocyte subset populations of their spleens. CD4+ lymphocytes were almost completely eliminated from the spleens of the anti-CD4 mAb-treated MRL/lpr mice, with a reduction in the CD4+ T cells from 18.1% in the saline-treated group and 20.8% in the rat Ig-treated group to 0.6% in the anti-CD4 mAb-treated group. Lymphoid hyperplasia, normally present in 5-month-old MRL/lpr mice, was markedly reduced by treatment with antiCD4 mAb, as shown in Table 1. Lymph node weights were reduced approximately 60% (P < 0.001, Student t test), while spleen weights were reduced 50 to 56% (P < 0.002) by treatment with anti-CD4 mAb. In both control groups histologic evaluation revealed complete replacement of normal nodal architecture by the massive accumulation of a homogeneous population of small lymphocytes. There was no change in the general histologic topography of the anti-CD4 mAb-treated mice compared to control animals, except for a diminution in the monotonous small lymphocyte areas between recognizable cortical zones. Renal pathology. Anti-CD4 mAb-treatment markedly reduced the renal pathology seen in MRL/lpr mice, as outlined in Table 2. Control treated MRL/lpr mice developed glomerulonephritis (Fig. I), vasculitis (Fig. 2) and an interstitial nephritis in the ductular region, changes previously described in untreated MRL/lpr mice (l-5). These lesions were largely abolished by treatment with anti-CD4 mAb (Figs. 3 and 4). Glomerulonephritis was detected in 9 of 9 saline-treated, 9 of 9 rat Ig-treated, and in only 1 of 9 anti-CD4 mAb-treated mice (P < 0.0 1, anti-CD4 mAb vs either control group, Fisher’s exact test). Renal vasculitis was detected in 6 of 9 saline-treated, 7 of 9 rat IgTABLE Effect of Anti-CD4
Treatment
of MRL/lpr Lymph
Treatment
group
Saline (9) Rat immunoglobulin Anti-CD4 (9)
(N)
(9)
I Mice
on Lymphoproliferation
node weights” k) 2.9 i 1.2” 3.0 k 0.8 1.2 * 0.5’
a Weight of submandibular, axillary. and inguinal nodes/animal. ’ Means k standard deviation. ’ Significantly different from either control at P < 0.00 1. d Significantly different from either control at P < 0.002.
Spleen weight k) 0.8 f 0.2 0.9 f 0.3 0.4 4 0.2d
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JABS ET AL. TABLE 2 Effect of Anti-CD4 Treatment of MRL/lpr
Treatment group Saline Rat immunoglobulin Anti-CD4
Mice on Renal Pathology
Vasculitis
Interstitial nephritis
Proteinuria > 1+n
9196 919
619 719
619 719
415 719
l/9<
O/9’
2/9d
2/9d
Glomerulonephritis
(2On a scale of 0 to 4+. b Number of animals with lesions/number of animals analyzed. ‘P < 0.01 and dP < 0.05 comparing anti-CD4 mAb treatment group to either control group.
treated, and 0 of 9 anti-CD4 mAb-treated mice (P < 0.01). Interstitial nephritis was detected in 6 of 9 saline-treated, 7 of 9 rat Ig-treated, but in only 2 of 9 anti-CD4 mAb-treated mice (P < 0.05). Proteinuria 3 1+ was detected in 4 of 5 saline-treated mice, 7 of 9 rat Ig-treated mice, and in only 2 of 9 anti-CD4 mAb-treated mice (P < 0.05). Autoantibodies and immunoglobulin levels. ANA were detected significantly more frequently in the control groups than in the anti-CD4 mAb-treated animals (salinetreated 7 of 7, rat Ig-treated 9 of 9, anti-CD4 mAb-treated 2 of 8, P < 0.01). Furthermore, among those animals with positive immunofluorescence, the staining was weaker
FIG. 1. Kidney from a saline-treated 5-month-old MRL/lpr toxylin and eosin, X 600).
mouse, showing glomerulonephritis
(hema-
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FIG. 2. Renal artery from a saline-treated 5-month-old MRL/lpr and eosin, X 400).
DISEASE
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mouse, showing vasculitis (hematoxylin
in those animals treated with anti-CD4 mAb than in the control groups. The mean grade (on a scale of 0 to 4+) was 2+ in the saline-treated, 1.6+ in the rat Ig-treated, and 1.3+ only in the two anti-CD4 mAb-treated mice with positive ANA; the remaining six anti-CD4 mAb-treated mice did not have ANA. Three different types of ANA patterns (homogeneous, peripheral, and speckled) were detected in the control groups, while only homogeneous and peripheral patterns were detected in the two anti-CD4 mAb-treated mice with ANA. Anti-dsDNA antibodies were significantly reduced by anti-CD4 mAb treatment. Compared to BALB/c and to 4-week-old MRL/lpr mice, Smonth-old MRL/lpr, 5month-old saline-treated MRL/lpr, and 5-month-old rat Ig-treated mice all had elevated titers of anti-dsDNA antibodies. Anti-CD4 mAb-treated mice had significantly reduced titers of anti-dsDNA antibodies (Fig. 5; P < 0.005, ANOVA). Analysis of the Ig class of the anti-dsDNA antibodies demonstrated that treatment with anti-CD4 mAb significantly reduced the level of IgG anti-dsDNA antibodies (Fig. 6; P < 0.05) to levels similar to those seen in 4-week-old MRL/lpr mice, while the low levels of IgM and IgA anti-dsDNA antibodies were not different among any of the treatment groups. Total serum IgG levels were also reduced by anti-CD4 mAb treatment (Fig. 7). Untreated 5-month-old MRL/lpr as well as control saline and rat Ig-treated animals all had elevated levels of Ig compared to l-month-old MRL/lpr mice. Anti-CD4 mAb treatment significantly reduced the levels of IgG (P < 0.05); levels of total IgM were similar among all groups. Levels of IgA were not significantly different among anti-
JABS ET AL.
FIG. 3. Kidney from an anti-CD4 mAb-treated 5-month-old MRL/lpr mouse, showing absence of disease (hematoxylin and eosin, X 490).
CD4 mAb-treated, l-month-old MRL/lpr, 5-month-old MRL/lpr, saline-treated, and rat Ig-treated mice, although there was a suggestion of lower levels in the l-monthold MRL/lpr and anti-CD4 mAb-treated mice. DISCUSSION MRL/lpr mice are congenic with MRL/+ mice and differ from them only by the presence of the autosomal recessive Zpr gene. This gene markedly accelerates the autoimmune disease in MRL mice and causes massive lymphadenopathy with lymph node weights up to 100 times normal (l-4). The primary immunologic disorder in MRL/lpr mice appears to be T cell mediated. Neonatal thymectomy of MRL/lpr mice ( 16, 17) or treatment of this substrain with monoclonal anti-T cell mAb (9) results in amelioration of the autoimmune disease. Similarly, the polyclonal B cell activation, hypergammaglobulinemia, and autoantibody formation in MRL/lpr mice appear to be T cell driven, since lymphocytes of MRL/lpr mice spontaneously secrete a B cell differentiation factor in vitro (18, 19). Among the many systemic lesions seen in MRL/ lpr mice is vasculitis (5, 20-22), which appears to be largely T cell mediated, with the preponderance of infiltrating lymphocytes being CD4+ T cells (5). A similar immunohistochemical profile is also seen in both the lacrimal and salivary gland lesions in these mice (5,23). The results of the present study show a dramatic reduction in both the incidence and severity of the autoimmune lesions, improvement in autoantibody
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FIG.4. Renal artery from an anti-CD4 mAb-treated 5-month-old MRL/lpr disease (hematoxylin and eosin, X 480).
503
mouse, showing absence of
production, and decrease in IgG levels in the group treated with anti-CD4 mAb, and are consistent with our hypothesis that the CD4’ T cell plays a central role in the development of the autoimmune disease in MRL/lpr mice. All aspects of the renal disease were markedly improved by treatment with antiCD4 mAb. Glomerulonephritis, interstitial nephritis, and proteinuria were either reduced or completely eliminated. Vasculitis was not detected in any of the anti-CD4 mAb-treated animals, but was present in over two-thirds of the control animals. Perivascular sheathing was present in all control animals, even those without true vasculitis. Mild sheathing without vasculitis was seen in only three of nine anti-CD4 mAb-treated animals. The elimination of vasculitis is consistent with our histologic finding that the majority of the infiltrating mononuclear inflammatory cells in the vascular walls are CD4+ (5). While we (5) and others (23) have found that the lacrimal gland, salivary gland, and renal vasculitic lesions are composed primarily of CD4+ T cells, Moyer et al. (22) reported that the vasculitis was largely composed of double-negative T cells, similar to the massively enlarged lymph nodes in these mice. While the difference between their immunocytochemical results and ours remains unexplained, the results of treatment with anti-CD4 mAb described in the present study strongly suggest that the CD4+ T cell plays an important role in the vasculitis and are consistent with our previous findings on immunocytochemistry (5, 24). We have also found that MRL/ lpr mice develop ocular inflammatory lesions, predominantly scleral vasculitis and choroiditis (24) which are also virtually eliminated by treatment with anti-CD4 mAb (25).
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Ant&DNA FIG. 5. Anti-dsDNA antibody levels in the sera of BALB/c mice, 4-week-old and 5-month-old MRL/lpr mice, and MRL/lpr mice receiving treatment with saline, rat Ig, or anti-CD4 mAb. Each group was comprised of 2 to 9 mice. The antibody titer was defined as the reciprocal of the serum dilution giving an optical density of 0.100. Group means are given, and the bars represent standard deviations. Values with an asterisk (*) are significantly different from the saline-treated animals at a value of P < 0.005.
Antinuclear antibodies, anti-dsDNA antibody titers, and the abnormally high immunoglobulin levels were all improved by treatment with anti-CD4 mAb. MRL/lpr mice demonstrate similar abnormalities of B cell function as do other autoimmune mice, in that polyclonal B cell activation, hypergammaglobulinemia, and autoantibody formation are all present. However, in MRL/lpr mice the B cell abnormalities appear to be secondary to T cell dysfunction. It has been demonstrated in vitro that the lymphocytes of MRL/lpr mice spontaneously secrete a B cell differentiation factor ( 18, 19) which has been taken as evidence for T cells driving the B cell abnormalities in these mice. Treatment with anti-T cell mAb reduces the anti-DNA mAb levels in MRL/lpr mice (9). Our results support this hypothesis: elimination of CD4+ T cells resulted in suppression of autoantibody formation and normalization of the elevated Ig levels. Anti-CD4 mAb-treated MRL/lpr mice had IgG levels similar to those seen in 4-week-old MRL/lpr mice, animals which do not yet have evidence of autoimmune disease. Thus, the CD4+ T cell appears to be involved in driving the B cell abnormalities as noted above, presumably through the secretion of stimulatory lymphokines. MRL/lpr mice also develop a massive lymphadenopathy not seen in the congenic MRL/+ substrain. These massively enlarged lymph nodes are composed primarily of Thy 1.2+ CD4- CD8- TCR (r//3’ double-negative T cells (6-8). The exact nature of these double-negative T cells and their relationship to the other autoimmune lesions seen in MRL/lpr mice are not clear. The marked reduction in lymph node and splenic weights (50-60%) detected in the anti-CD4 mAb-treated mice suggests that the doublenegative T cell lymphoproliferation depends, at least in part, upon CD4+ T cells. A
ANTI-CD4
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0.4
H
MRUlpr-lmo
q q q
MRUlpf-ma
0
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Saline
Rat Ig
T
w
w AntidsDNA
FIG. 6. Subclass of anti-dsDNA antibodies in the sera of 4-week-old and 5-month-old MRL/lpr mice, and 5-month-old MRL/lpr mice receiving saline, rat Ig, or anti-CD4 mAb therapy. Antibody titers were tested by ELISA at a dilution of 1:2. Units are optical density, and group means are given. The bars represent standard deviations. Values with an asterisk (*) are significantly different from the saline-treated animals at a value of P < 0.05.
similar result was also reported by Santoro et al. (11) in their study, where spleen and lymph node weights were reduced 75 to 85% by anti-CD4 mAb treatment. In their study, animals were sacrificed at 14 weeks of age, while in our experiments at 20 weeks of age. The longer duration of treatment and older age at sacrifice in the present study were necessary to adequately test the hypothesis that anti-CD4 mAb therapy would eliminate the histologic evidence of autoimmune disease, an issue not previously addressed, as well as the lymphoproliferation and autoantibody formation. While the role of the double-negative T cell in the production of autoimmune disease in MRL/lpr mice is unclear, the lpr gene is felt to be a single autosomal recessive gene which both accelerates the autoimmune disease and produces the massive lymphadenopathy (3). It is possible that the two effects are independent and that the autoimmune disease is unrelated to the double-negative T cells, or that the two events are related and that the double-negative T cells play an essential role in the autoimmune disease. Since the double-negative T cells are Thy 1.2+, treatment with mAb to Thy 1.2 cannot distinguish between these two possibilities. Based upon our previous histologic results (5) we felt that the CD4+ T cell played an essential role in the autoimmune process and that the massive accumulation of double-negative T cells in the lymph nodes was not a primary factor in the development of the autoimmune disease. The results of the present study using anti-CD4 mAb therapy to treat MRL/lpr mice support this view. While the relationships among CD4+ T cells, double-negative T cells, and autoimmune lesions remain incompletely explained, it would appear that the double-negative T cells are not directly or solely responsible for the autoimmune disease in MRL/lpr
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n q q El Cl
MRUlpr-1
mo
MRt./lpr-5mo Saline Rat Ig Anti-CD4
Total lmmunogkhulin FIG. 7. Serum Ig levels for I- and 5-month-old MRL/lpr mice, and 5-month-old MRL/lpr mice receiving saline, rat Ig, or anti-CD4 mAb therapy. Antibody titers were tested by ELISA at dilution of 1:100,000 for total IgG and 1:20 for total IgA and IgM. Units are optical density, and group means are given. The bars represent standard deviations. Values with an asterisk (*) are significantly different from the saline-treated animals at a value of P < 0.05.
mice. The results of our experiments support the hypothesis that the CD4+ T cell plays a central role in the pathogenesis of autoimmune disease in this strain. REFERENCES 1. Andrews, B. S., Eisenberg, R. A., Theofilopoulos, A. N., Izui, S., Wilson, C. B., McConahey, P. J., Murphy, E. D., Roths, J. B., and Dixon, F. J., J. Exp. Med. 148, 1198, 1978. 2. Theofilopoulos, A. N., and Dixon, F. J., Immunol. Rev. 55, 179, 1981. 3. Murphy, E. D., In “Immunologic Defects in Laboratory Animals” (M. E. Gershwin and B. Merchant, Eds.), p. 143. Plenum Press, New York, 198 1. 4. Theofilopoulos, A. N., and Dixon, F. J., In “Advances in Immunology” (F. Dixon, Ed.), Vol. 37, p, 269. Academic Press, San Diego, CA, 1985. 5. Jabs, D. A., and Prendergast, R. A., J. hp. Med. 166, 1198, 1987. 6. Theofilopoulos, A. N., Eisenberg, R. A., Bourdon, M., Crowel, J. S., Jr., and Dixon, F. J., J. Exp. Med. 149, 5 16, 1979. 7. Lewis, D. E., Giorgi, J. V., and Warner, N. L., Nature 289, 298, 1981. 8. Wofsy, D., Hardy, R. R., and Seaman, W. E., J. Immunol. 132,2686, 1984. 9. Wofsy, D., Ledbetter, J. A., Hendler, P. L., and Seaman, W. E., J. Immunol. 134, 852, 1985. 10. Wofsy, D., and Seaman, W. E., J. Exp. Med. 161, 378, 1985. I I. Santoro, T. J., Portanova, J. P., and Kotzin, B. L., J. Exp. Med. 167, 1713, 1988. 12. Dialynas, D. P., Quan, Z. S., Wall, K. A., Quintans, J. M., Loken, R., Pierres, M., and Fitch, F. W., J. Immunol.
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13. Dialynas, D. P., Wilde, D. B., Marrack, P., Pierres, A. K., Wall, A., Havran, W., Hen, G. O., Loken, M. R., Pierres, M., Kappler, J., and Fitch, F. W., Immunol. Rev. 74, 29, 1983.
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14. Burek, C. L., and Rose, N. R., In “Diagnostic Immunopathology” (R. B. Colvin, A. K. Bahn, and R. T. McCluskey, Eds.), p. 87, Raven Press, New York, NY, 1988. 15. Pisetsky, D. S., and Peters, D. V., J. Immunol. Methods 41, 187, 1981. 16. Steinberg, A. D., Roths, J. B., Murphy, E. D., Steinberg, R. T., and Raveche. E. S., J. Immunol. 125, 871, 1980. 17. Hang, L., Theolilopoulos, A. N., Balderas, R. S., Francis, S. J.. and Dixon, F. J., J. Immune/. 132, 1809, 1984. 18. Prud’homme, G. J.. Park. C. L., Fieser. T. M., Kofler, R.. Dixon, F. J., and Theofilopoulos. A. N.. .I. Exp. Med. 157, 730, 1983. 19. Prud’homme, G. J., Balderas, R. S., Dixon, F. J., and Theofilopoulos, A. N.. J. E.x~. Med. 157, I8 15. 1983. 20. Berden, J. H. M., Hang, L., McConahey, P. J., and Dixon, F. J., J. Immunol. 130, 1699, 1983. 2 I. Alexander, E. L.. Moyer, C., Travlos, G. S.. Roths, J. B., and Murphy, E. D., Arthritis Rhetrm. 28, I 146. 1985. 22. Moyer, C. F.. Strandberg, J. D., and Reinisch, C. L., Am. J. Pathol. 127, 229, 1987. 23. Jonsson, R., Tarkeroski, A., Backman, K.. Holmdahl, R., and Klareskog. L., Immunology 60, 61 I. 1987. 24. Jabs, D. A., and Prendergast, R. A., Invest. Ophthalmol. Visual Ski. 32, 1944, 199 1. 25. Jabs. D. A., and Prendergast, R. A., Inwsst. Ophthalmol. Visual Sci. 32, 27 18, 199 1.
IMMUNOLOGY
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(1992)
Anti-CD4 Monoclonal Antibody Therapy Suppresses Autoimmune Disease in MRL/Mp-lpr/lpr Mice’ DOUGLASA.JABS,*,~ C.LYNNE BUREK,? QILE Hu,? RUDOLFC.KUPPERS,~ BELLALEE,* ANDROBERTA.PRENDERGAST* *The Wilmer Ophthalmological Institute, The Johns Hopkins University School of Medicine; and tthe Department of Immunology and Infectious Diseases, The Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205 Received December 3, 1991; accepted January 16, I992 MRL/Mp-lpr/lpr (MRL/lpr) mice spontaneously develop systemic autoimmune disease, characterized by vasculitis, lymphadenopathy, glomerulonephritis, and autoantibody formation. The target organ inflammatory lesions are composed largely of CD4+ “helper” T cells, while the massivelyenlarged lymph nodes are composed primarily of CD3+ CD4- CD8- TCR a//3’ “doublenegative” T cells. In this study we investigated the effect of treatment of MRL/lpr mice with antiCD4 monoclonal antibody (mAb); control groups consisted of animals treated with normal saline or rat immunoglobulin (Ig). Anti-CD4 mAb treatment, which was started at 4 weeks and continued through 20 weeks of age, resulted in a dramatic reduction of both the frequency and severity of the autoimmune disease, as demonstrated histologically and serologically. Anti-CD4 mAb therapy markedly reduced the frequency of glomerulonephritis and eliminated vasculitis of the major renal arterial branches. Glomerulonephritis was detected in 9 of 9 saline-treated, 9 of 9 rat Igtreated, but in only 1 of 9 anti-CD4 mAb-treated mice; vasculitis was detected in 6 of 9 salinetreated, 7 of 9 rat Ig-treated, but in none of 9xanti-CD4 mAb-treated mice. The frequency of antinuclear antibodies, titer of anti-dsDNA antibodies, and total Ig levels were all significantly reduced by anti-CD4 mAb therapy. These data support the hypothesis that CD4+ T cells play a central role in the disease process in this autoimmune strain. 0 1992 Academic PXSS. I~C.
INTRODUCTION Several strains of autoimmune mice have been described, including MRL/Mp-lpr/ lpr (MRL/lpr)3, MRL/Mp-+/+ (MRL/+), (NZBxNZW) Fl hybrid (NZB/W), BXSB, and Palmerston-North strains (l-4). MRL/lpr spontaneously develop an autoimmune disease characterized by vasculitis, massive lymphadenopathy, glomerulonephritis, and autoantibody formation. The target organ lesions, including vasculitis, are largely ’ This study was supported in part by Grants EY05912, EY03521, EY01765, and AR31632 from the National Institutes of Health, Bethesda, Maryland, and an unrestricted grant from Research to Prevent Blindness. Dr. Jabs is a Research to Prevent Blindness Olga Keith Wiess Scholar. 2 To whom correspondence should be addressed at The Wilmer Ophthalmological Institute, 550 North Broadway, Suite 700, Baltimore, MD 2 1205. 3 Abbreviations used: MRL/lpr, MRL/Mp-lpr/lpr; MRL/+, MRL/Mp-+/+; NZBJW, (NZB X NZW) Fl hybrid; mAb, monoclonal antibody; SCID, severe combined immunodeficiency; Ig, immunoglobulin; FITC, fluorescein isothiocyanate; ANA, antinuclear antibodies; AP, alkaline phosphatase. 496 000%8749192 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form resewed
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composed of CD4+ T cells (5) while the massively enlarged lymph nodes are composed primarily of Thy 1.2+ CD4- CD8- TCR a/P’ “double-negative” T cells (5-8). A previous report by Wofsy et al. (9) has underscored the importance of T cells in the development of disease in MRL/lpr mice, where the autoimmune disease was suppressed by treatment with the anti-T cell monoclonal antibody (mAb), anti-Thy 1.2, as evidenced by decreased autoantibody production, decreased proteinuria, and improved survival. This result was different from that seen in NZB/W mice, where antiT cell mAb treatment did not alter the disease, or in BXSB mice, where such treatment resulted in fatal anaphylaxis (9). While this study emphasized the important role that T cells play in the disease in MRL/lpr mice, it did not distinguish between an effect on CD4+ T cells or on double-negative T cells. Anti-CD4 mAb has been used to treat NZB/W mice and resulted in suppression of their autoimmune disease (10). The discrepancy between the results of treatment with anti-T cell mAb and anti-CD4 mAb in NZB/W mice remains unexplained. Our previous investigation of the vasculitic lesions in the kidneys of MRL/lpr mice revealed that the infiltrating mononuclear cells were primarily T cells, of which 58% were CD4+ and 12% CD8+, together with 5% B cells and 3% macrophages; a similar immunohistologic profile was found in the lacrimal gland lesions (5). This result suggested to us that the beneficial effect of treatment with anti-T cell mAb on the autoimmune lesions in MRL/lpr mice might be due to an effect upon the CD4’ T cell. In addition, anti-CD4 mAb was recently found to suppress autoantibody production and lymphadenopathy in MRL/lpr mice (11). The present study using anti-CD4 mAb therapy demonstrates marked suppression of the autoimmune disease as determined by reversal of the immunopathologic lesions, as well as the serologic abnormalities normally present in older MRL/lpr mice. MATERIALS
AND METHODS
Monoclonal antibodies. Monoclonal antibodies were prepared using the method of Wofsy et al. (9, 10). Hybridoma cells secreting a mAb to the murine CD4 antigen L3T4 (GK 1.5) were obtained from ATCC (Rockville, MD) (12, 13). One million cells were injected into the peritoneal cavity of severe combined immunodeficiency (SCID) mice obtained from Jackson Laboratories (Bar Harbor, ME). Antibody was harvested from the SCID peritoneal exudate by paracentesis starting 8 days after cell transfer. lmmunoglobulin (lg) was partially purified using ammonium sulfate precipitation followed by solubilization and dialysis against phosphate-buffered saline (PBS). The partially purified mAb was then standardized for protein content using spectrophotometric absorbance at 280 nm. Treatment protocol. MRL/lpr mice were obtained from the Jackson Laboratories and kept under standard conditions in the animal facilities of the Woods Research Building of The Johns Hopkins Hospital. Animals were treated starting at 1 month of age with one initial intravenous and one intraperitoneal injection of 2 mg each followed by weekly intraperitoneal injections of 2 mg of anti-L3T4 mAb. Two control groups were used: one was given injections of normal saline, and a second was given injections of rat lg. The rat lg used in these experiments was produced by subjecting normal rat serum to an ammonium sulfate precipitation and dialysis identical to that used for the preparation of the monoclonal anti-L3T4 mAb. At 5 months of age,
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animals were sacrificed by exsanguination. Sera were collected for the analyses outlined below. Proteinuria was measured using dipstick analysis, which is graded in a semiquantitative scale of 0 to 4f (Albustix; Miles Inc., Sebhart, IN). Proteinuria was classified as normal if it was graded 0 or trace and abnormal if it was l+ or greater. Submandibular, axillary, and inguinal nodes were removed and weighed to give a measurement of the lymphadenopathy. Spleens were removed and weighed separately. Kidneys were removed for histologic processing, fixed in 4% buffered formaldehyde, embedded in paraffin, sectioned at 5 pm, and stained with hematoxylin and eosin. Histopathologic evaluation was performed by a masked reader (R.A.P.) unaware of the treatment for each animal. Lymphocyte subset analysis. Animals were sacrificed at 3 months of age for flow cytometry analysis of lymphocyte subset populations of their spleens. Spleens were removed aseptically; a cell suspension isolated; and 4 X 1O6spleen cells were suspended in 1 ml PBS and incubated for 30 min with the primary antibody. Antibodies used included anti-Thy 1.2 (Becton-Dickinson, Mountain View, CA) for T cells, anti-L3T4 (Becton-Dickinson) for CD4+ T cells, and anti-Lyt 2 (Becton-Dickinson) for CD8+ T cells. Cells were washed, resuspended, and incubated for 30 min with a fluorescein isothiocyanate (FITC)-conjugated goat anti-rat antibody (Organon Tekika, West Chester, PA). The percentage of cells staining positively was enumerated using a Facscan flow cytometer (Becton-Dickinson). Negative controls for each analysis used rat Ig for the primary antibody. Antinuclear antibodies. Antinuclear antibodies (ANA) were measured using indirect immunofluorescence. Sera were screened at a 1: 10 dilution, using 4-pm frozen sections of rat liver as the substrate and a FITC-conjugated goat anti-mouse mAb as the second antibody. ANA were read in a masked fashion and scored on a semiquantitative scale score of 0 to 4+ as previously described (14) where 0 is negative, 1+ is weak, 2+ is medium, 3+ is strong, and 4+ is very strong staining. Measurement of anti-DNA antibodies. Determination of anti-double-stranded DNA (anti-dsDNA) antibodies was performed using the method described by Pisetsky and Peters (15). Briefly, ELISA plates (Corning Lab Sciences Co., New York, NY) were coated with 200 ~1 of salmon testes DNA (Sigma, St. Louis, MO, 5 pg/ml in PBS) in each well and incubated at 37°C for 2-3 hr. After decanting and washing the plates three times with PBS-Tween 20, the remaining sites in each well were blocked by incubation with 200 ~1 of 1 mg/ml methylated bovine serum albumin at 25°C for 1 hr. Plates were washed three times with PBS-Tween 20; then serum diluted 1:2 with PBS-Tween 20 was added and the plates were incubated at 25°C for 1 hr. At the end of the incubation, plates were washed three times and 100 ~1 of 1: 1000 dilution of alkaline phosphatase (AP)-coupled goat anti-mouse Ig (whole molecule) or, for subclass determination, goat anti-mouse IgM, IgG, or IgA AP-conjugate (Sigma) was added to each well. After 1-hr incubation at 25°C each well was again washed three times, and then p-phenol phosphate was added as a substrate. Reactions were stopped with the addition of 50 ~1 of 3 M NaOH in each well for 10 min. Each serum was also tested on plates without coating with DNA. Sera from BALB/c and 4-week-old MRL/lpr mice were used as negative controls, while sera from untreated 5-month-old MRL/ lpr mice were used as positive controls. Plates were read using optical density at 410 nm. Autoantibody determinations were performed in a masked fashion.
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Determination of total immunoglobulin. ELISA plates were coated with 100 ~1 of 5 pg/ml goat anti-mouse Ig (IgM, IgG, IgA; Capell, Cochranville, PA) in each well at 4°C overnight. Plates were blocked with 1% BSA; after washing the plates three times with PBS-Tween 20, 100 ~1 of serum at dilutions of 1:2 to 1: 100,000 were added to the individual wells and incubated at 25°C for 1 hr. Relative antibody subclass concentrations were determined using AP-conjugated goat anti-mouse IgG, IgM, or IgA as described above. RESULTS Lymphoprolijkation. Two animals from each group (saline, rat Ig, anti-CD4 mAb treatment) were sacrificed at 3 months of age for analysis of the lymphocyte subset populations of their spleens. CD4+ lymphocytes were almost completely eliminated from the spleens of the anti-CD4 mAb-treated MRL/lpr mice, with a reduction in the CD4+ T cells from 18.1% in the saline-treated group and 20.8% in the rat Ig-treated group to 0.6% in the anti-CD4 mAb-treated group. Lymphoid hyperplasia, normally present in 5-month-old MRL/lpr mice, was markedly reduced by treatment with antiCD4 mAb, as shown in Table 1. Lymph node weights were reduced approximately 60% (P < 0.001, Student t test), while spleen weights were reduced 50 to 56% (P < 0.002) by treatment with anti-CD4 mAb. In both control groups histologic evaluation revealed complete replacement of normal nodal architecture by the massive accumulation of a homogeneous population of small lymphocytes. There was no change in the general histologic topography of the anti-CD4 mAb-treated mice compared to control animals, except for a diminution in the monotonous small lymphocyte areas between recognizable cortical zones. Renal pathology. Anti-CD4 mAb-treatment markedly reduced the renal pathology seen in MRL/lpr mice, as outlined in Table 2. Control treated MRL/lpr mice developed glomerulonephritis (Fig. I), vasculitis (Fig. 2) and an interstitial nephritis in the ductular region, changes previously described in untreated MRL/lpr mice (l-5). These lesions were largely abolished by treatment with anti-CD4 mAb (Figs. 3 and 4). Glomerulonephritis was detected in 9 of 9 saline-treated, 9 of 9 rat Ig-treated, and in only 1 of 9 anti-CD4 mAb-treated mice (P < 0.0 1, anti-CD4 mAb vs either control group, Fisher’s exact test). Renal vasculitis was detected in 6 of 9 saline-treated, 7 of 9 rat IgTABLE Effect of Anti-CD4
Treatment
of MRL/lpr Lymph
Treatment
group
Saline (9) Rat immunoglobulin Anti-CD4 (9)
(N)
(9)
I Mice
on Lymphoproliferation
node weights” k) 2.9 i 1.2” 3.0 k 0.8 1.2 * 0.5’
a Weight of submandibular, axillary. and inguinal nodes/animal. ’ Means k standard deviation. ’ Significantly different from either control at P < 0.00 1. d Significantly different from either control at P < 0.002.
Spleen weight k) 0.8 f 0.2 0.9 f 0.3 0.4 4 0.2d
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JABS ET AL. TABLE 2 Effect of Anti-CD4 Treatment of MRL/lpr
Treatment group Saline Rat immunoglobulin Anti-CD4
Mice on Renal Pathology
Vasculitis
Interstitial nephritis
Proteinuria > 1+n
9196 919
619 719
619 719
415 719
l/9<
O/9’
2/9d
2/9d
Glomerulonephritis
(2On a scale of 0 to 4+. b Number of animals with lesions/number of animals analyzed. ‘P < 0.01 and dP < 0.05 comparing anti-CD4 mAb treatment group to either control group.
treated, and 0 of 9 anti-CD4 mAb-treated mice (P < 0.01). Interstitial nephritis was detected in 6 of 9 saline-treated, 7 of 9 rat Ig-treated, but in only 2 of 9 anti-CD4 mAb-treated mice (P < 0.05). Proteinuria 3 1+ was detected in 4 of 5 saline-treated mice, 7 of 9 rat Ig-treated mice, and in only 2 of 9 anti-CD4 mAb-treated mice (P < 0.05). Autoantibodies and immunoglobulin levels. ANA were detected significantly more frequently in the control groups than in the anti-CD4 mAb-treated animals (salinetreated 7 of 7, rat Ig-treated 9 of 9, anti-CD4 mAb-treated 2 of 8, P < 0.01). Furthermore, among those animals with positive immunofluorescence, the staining was weaker
FIG. 1. Kidney from a saline-treated 5-month-old MRL/lpr toxylin and eosin, X 600).
mouse, showing glomerulonephritis
(hema-
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FIG. 2. Renal artery from a saline-treated 5-month-old MRL/lpr and eosin, X 400).
DISEASE
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mouse, showing vasculitis (hematoxylin
in those animals treated with anti-CD4 mAb than in the control groups. The mean grade (on a scale of 0 to 4+) was 2+ in the saline-treated, 1.6+ in the rat Ig-treated, and 1.3+ only in the two anti-CD4 mAb-treated mice with positive ANA; the remaining six anti-CD4 mAb-treated mice did not have ANA. Three different types of ANA patterns (homogeneous, peripheral, and speckled) were detected in the control groups, while only homogeneous and peripheral patterns were detected in the two anti-CD4 mAb-treated mice with ANA. Anti-dsDNA antibodies were significantly reduced by anti-CD4 mAb treatment. Compared to BALB/c and to 4-week-old MRL/lpr mice, Smonth-old MRL/lpr, 5month-old saline-treated MRL/lpr, and 5-month-old rat Ig-treated mice all had elevated titers of anti-dsDNA antibodies. Anti-CD4 mAb-treated mice had significantly reduced titers of anti-dsDNA antibodies (Fig. 5; P < 0.005, ANOVA). Analysis of the Ig class of the anti-dsDNA antibodies demonstrated that treatment with anti-CD4 mAb significantly reduced the level of IgG anti-dsDNA antibodies (Fig. 6; P < 0.05) to levels similar to those seen in 4-week-old MRL/lpr mice, while the low levels of IgM and IgA anti-dsDNA antibodies were not different among any of the treatment groups. Total serum IgG levels were also reduced by anti-CD4 mAb treatment (Fig. 7). Untreated 5-month-old MRL/lpr as well as control saline and rat Ig-treated animals all had elevated levels of Ig compared to l-month-old MRL/lpr mice. Anti-CD4 mAb treatment significantly reduced the levels of IgG (P < 0.05); levels of total IgM were similar among all groups. Levels of IgA were not significantly different among anti-
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FIG. 3. Kidney from an anti-CD4 mAb-treated 5-month-old MRL/lpr mouse, showing absence of disease (hematoxylin and eosin, X 490).
CD4 mAb-treated, l-month-old MRL/lpr, 5-month-old MRL/lpr, saline-treated, and rat Ig-treated mice, although there was a suggestion of lower levels in the l-monthold MRL/lpr and anti-CD4 mAb-treated mice. DISCUSSION MRL/lpr mice are congenic with MRL/+ mice and differ from them only by the presence of the autosomal recessive Zpr gene. This gene markedly accelerates the autoimmune disease in MRL mice and causes massive lymphadenopathy with lymph node weights up to 100 times normal (l-4). The primary immunologic disorder in MRL/lpr mice appears to be T cell mediated. Neonatal thymectomy of MRL/lpr mice ( 16, 17) or treatment of this substrain with monoclonal anti-T cell mAb (9) results in amelioration of the autoimmune disease. Similarly, the polyclonal B cell activation, hypergammaglobulinemia, and autoantibody formation in MRL/lpr mice appear to be T cell driven, since lymphocytes of MRL/lpr mice spontaneously secrete a B cell differentiation factor in vitro (18, 19). Among the many systemic lesions seen in MRL/ lpr mice is vasculitis (5, 20-22), which appears to be largely T cell mediated, with the preponderance of infiltrating lymphocytes being CD4+ T cells (5). A similar immunohistochemical profile is also seen in both the lacrimal and salivary gland lesions in these mice (5,23). The results of the present study show a dramatic reduction in both the incidence and severity of the autoimmune lesions, improvement in autoantibody
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FIG.4. Renal artery from an anti-CD4 mAb-treated 5-month-old MRL/lpr disease (hematoxylin and eosin, X 480).
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mouse, showing absence of
production, and decrease in IgG levels in the group treated with anti-CD4 mAb, and are consistent with our hypothesis that the CD4’ T cell plays a central role in the development of the autoimmune disease in MRL/lpr mice. All aspects of the renal disease were markedly improved by treatment with antiCD4 mAb. Glomerulonephritis, interstitial nephritis, and proteinuria were either reduced or completely eliminated. Vasculitis was not detected in any of the anti-CD4 mAb-treated animals, but was present in over two-thirds of the control animals. Perivascular sheathing was present in all control animals, even those without true vasculitis. Mild sheathing without vasculitis was seen in only three of nine anti-CD4 mAb-treated animals. The elimination of vasculitis is consistent with our histologic finding that the majority of the infiltrating mononuclear inflammatory cells in the vascular walls are CD4+ (5). While we (5) and others (23) have found that the lacrimal gland, salivary gland, and renal vasculitic lesions are composed primarily of CD4+ T cells, Moyer et al. (22) reported that the vasculitis was largely composed of double-negative T cells, similar to the massively enlarged lymph nodes in these mice. While the difference between their immunocytochemical results and ours remains unexplained, the results of treatment with anti-CD4 mAb described in the present study strongly suggest that the CD4+ T cell plays an important role in the vasculitis and are consistent with our previous findings on immunocytochemistry (5, 24). We have also found that MRL/ lpr mice develop ocular inflammatory lesions, predominantly scleral vasculitis and choroiditis (24) which are also virtually eliminated by treatment with anti-CD4 mAb (25).
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Ant&DNA FIG. 5. Anti-dsDNA antibody levels in the sera of BALB/c mice, 4-week-old and 5-month-old MRL/lpr mice, and MRL/lpr mice receiving treatment with saline, rat Ig, or anti-CD4 mAb. Each group was comprised of 2 to 9 mice. The antibody titer was defined as the reciprocal of the serum dilution giving an optical density of 0.100. Group means are given, and the bars represent standard deviations. Values with an asterisk (*) are significantly different from the saline-treated animals at a value of P < 0.005.
Antinuclear antibodies, anti-dsDNA antibody titers, and the abnormally high immunoglobulin levels were all improved by treatment with anti-CD4 mAb. MRL/lpr mice demonstrate similar abnormalities of B cell function as do other autoimmune mice, in that polyclonal B cell activation, hypergammaglobulinemia, and autoantibody formation are all present. However, in MRL/lpr mice the B cell abnormalities appear to be secondary to T cell dysfunction. It has been demonstrated in vitro that the lymphocytes of MRL/lpr mice spontaneously secrete a B cell differentiation factor ( 18, 19) which has been taken as evidence for T cells driving the B cell abnormalities in these mice. Treatment with anti-T cell mAb reduces the anti-DNA mAb levels in MRL/lpr mice (9). Our results support this hypothesis: elimination of CD4+ T cells resulted in suppression of autoantibody formation and normalization of the elevated Ig levels. Anti-CD4 mAb-treated MRL/lpr mice had IgG levels similar to those seen in 4-week-old MRL/lpr mice, animals which do not yet have evidence of autoimmune disease. Thus, the CD4+ T cell appears to be involved in driving the B cell abnormalities as noted above, presumably through the secretion of stimulatory lymphokines. MRL/lpr mice also develop a massive lymphadenopathy not seen in the congenic MRL/+ substrain. These massively enlarged lymph nodes are composed primarily of Thy 1.2+ CD4- CD8- TCR (r//3’ double-negative T cells (6-8). The exact nature of these double-negative T cells and their relationship to the other autoimmune lesions seen in MRL/lpr mice are not clear. The marked reduction in lymph node and splenic weights (50-60%) detected in the anti-CD4 mAb-treated mice suggests that the doublenegative T cell lymphoproliferation depends, at least in part, upon CD4+ T cells. A
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0.4
H
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q q q
MRUlpf-ma
0
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Rat Ig
T
w
w AntidsDNA
FIG. 6. Subclass of anti-dsDNA antibodies in the sera of 4-week-old and 5-month-old MRL/lpr mice, and 5-month-old MRL/lpr mice receiving saline, rat Ig, or anti-CD4 mAb therapy. Antibody titers were tested by ELISA at a dilution of 1:2. Units are optical density, and group means are given. The bars represent standard deviations. Values with an asterisk (*) are significantly different from the saline-treated animals at a value of P < 0.05.
similar result was also reported by Santoro et al. (11) in their study, where spleen and lymph node weights were reduced 75 to 85% by anti-CD4 mAb treatment. In their study, animals were sacrificed at 14 weeks of age, while in our experiments at 20 weeks of age. The longer duration of treatment and older age at sacrifice in the present study were necessary to adequately test the hypothesis that anti-CD4 mAb therapy would eliminate the histologic evidence of autoimmune disease, an issue not previously addressed, as well as the lymphoproliferation and autoantibody formation. While the role of the double-negative T cell in the production of autoimmune disease in MRL/lpr mice is unclear, the lpr gene is felt to be a single autosomal recessive gene which both accelerates the autoimmune disease and produces the massive lymphadenopathy (3). It is possible that the two effects are independent and that the autoimmune disease is unrelated to the double-negative T cells, or that the two events are related and that the double-negative T cells play an essential role in the autoimmune disease. Since the double-negative T cells are Thy 1.2+, treatment with mAb to Thy 1.2 cannot distinguish between these two possibilities. Based upon our previous histologic results (5) we felt that the CD4+ T cell played an essential role in the autoimmune process and that the massive accumulation of double-negative T cells in the lymph nodes was not a primary factor in the development of the autoimmune disease. The results of the present study using anti-CD4 mAb therapy to treat MRL/lpr mice support this view. While the relationships among CD4+ T cells, double-negative T cells, and autoimmune lesions remain incompletely explained, it would appear that the double-negative T cells are not directly or solely responsible for the autoimmune disease in MRL/lpr
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n q q El Cl
MRUlpr-1
mo
MRt./lpr-5mo Saline Rat Ig Anti-CD4
Total lmmunogkhulin FIG. 7. Serum Ig levels for I- and 5-month-old MRL/lpr mice, and 5-month-old MRL/lpr mice receiving saline, rat Ig, or anti-CD4 mAb therapy. Antibody titers were tested by ELISA at dilution of 1:100,000 for total IgG and 1:20 for total IgA and IgM. Units are optical density, and group means are given. The bars represent standard deviations. Values with an asterisk (*) are significantly different from the saline-treated animals at a value of P < 0.05.
mice. The results of our experiments support the hypothesis that the CD4+ T cell plays a central role in the pathogenesis of autoimmune disease in this strain. REFERENCES 1. Andrews, B. S., Eisenberg, R. A., Theofilopoulos, A. N., Izui, S., Wilson, C. B., McConahey, P. J., Murphy, E. D., Roths, J. B., and Dixon, F. J., J. Exp. Med. 148, 1198, 1978. 2. Theofilopoulos, A. N., and Dixon, F. J., Immunol. Rev. 55, 179, 1981. 3. Murphy, E. D., In “Immunologic Defects in Laboratory Animals” (M. E. Gershwin and B. Merchant, Eds.), p. 143. Plenum Press, New York, 198 1. 4. Theofilopoulos, A. N., and Dixon, F. J., In “Advances in Immunology” (F. Dixon, Ed.), Vol. 37, p, 269. Academic Press, San Diego, CA, 1985. 5. Jabs, D. A., and Prendergast, R. A., J. hp. Med. 166, 1198, 1987. 6. Theofilopoulos, A. N., Eisenberg, R. A., Bourdon, M., Crowel, J. S., Jr., and Dixon, F. J., J. Exp. Med. 149, 5 16, 1979. 7. Lewis, D. E., Giorgi, J. V., and Warner, N. L., Nature 289, 298, 1981. 8. Wofsy, D., Hardy, R. R., and Seaman, W. E., J. Immunol. 132,2686, 1984. 9. Wofsy, D., Ledbetter, J. A., Hendler, P. L., and Seaman, W. E., J. Immunol. 134, 852, 1985. 10. Wofsy, D., and Seaman, W. E., J. Exp. Med. 161, 378, 1985. I I. Santoro, T. J., Portanova, J. P., and Kotzin, B. L., J. Exp. Med. 167, 1713, 1988. 12. Dialynas, D. P., Quan, Z. S., Wall, K. A., Quintans, J. M., Loken, R., Pierres, M., and Fitch, F. W., J. Immunol.
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14. Burek, C. L., and Rose, N. R., In “Diagnostic Immunopathology” (R. B. Colvin, A. K. Bahn, and R. T. McCluskey, Eds.), p. 87, Raven Press, New York, NY, 1988. 15. Pisetsky, D. S., and Peters, D. V., J. Immunol. Methods 41, 187, 1981. 16. Steinberg, A. D., Roths, J. B., Murphy, E. D., Steinberg, R. T., and Raveche. E. S., J. Immunol. 125, 871, 1980. 17. Hang, L., Theolilopoulos, A. N., Balderas, R. S., Francis, S. J.. and Dixon, F. J., J. Immune/. 132, 1809, 1984. 18. Prud’homme, G. J.. Park. C. L., Fieser. T. M., Kofler, R.. Dixon, F. J., and Theofilopoulos. A. N.. .I. Exp. Med. 157, 730, 1983. 19. Prud’homme, G. J., Balderas, R. S., Dixon, F. J., and Theofilopoulos, A. N.. J. E.x~. Med. 157, I8 15. 1983. 20. Berden, J. H. M., Hang, L., McConahey, P. J., and Dixon, F. J., J. Immunol. 130, 1699, 1983. 2 I. Alexander, E. L.. Moyer, C., Travlos, G. S.. Roths, J. B., and Murphy, E. D., Arthritis Rhetrm. 28, I 146. 1985. 22. Moyer, C. F.. Strandberg, J. D., and Reinisch, C. L., Am. J. Pathol. 127, 229, 1987. 23. Jonsson, R., Tarkeroski, A., Backman, K.. Holmdahl, R., and Klareskog. L., Immunology 60, 61 I. 1987. 24. Jabs, D. A., and Prendergast, R. A., Invest. Ophthalmol. Visual Ski. 32, 1944, 199 1. 25. Jabs. D. A., and Prendergast, R. A., Inwsst. Ophthalmol. Visual Sci. 32, 27 18, 199 1.