Suppression of systemic lupus erythematosus disease in mice by oral administration of kidney extract.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the increased production of antibodies reactive with a variety of self and non-self antigens. A number of immunomodulatory therapies have been investigated for the disease process. Intragastric administration of low dose kidney extract (KE) three times weekly for 5 weeks and then weekly until 6 months of age in SLE mice, showed decreased anti-dsDNA antibody levels, less kidney damage and significantly prolonged survival compared with control phosphate buffered saline (PBS)-fed mice. The KE-fed mice also exhibited reduced T cell proliferative response to KE in comparison with PBS-fed controls. Serum isotype distribution of the anti-dsDNA antibodies revealed a marked reduction of IgG1 and IgG3 responses in the KE-fed mice. While the renal inflammatory cell infiltration and expression of interleukin-4 (IL-4) and IL-10 were markedly suppressed, no local enhancement of transforming growth factor-beta (TGF-beta) was detected. Oral administration of low dose KE, however, upregulated expression of IL-2, IFN-gamma and TNF-alpha in the kidneys and suppressed glomerulonephritis. These findings suggest that oral KE affects the disease process in SLE and raise the possibility that oral administration of KE or other potential autoantigens may provide a new approach for the treatment of SLE.

Induction of systemic lupus erythematosus-like disease in mice by immunization with heparan sulfate.

Experimental systemic lupus erythematosus (SLE)-like disease was induced in BALB/c mice by immunization with heparan sulfate, the major glycosaminoglycan of glomerular basement membrane. Following booster injections with heparan sulfate (HS), high levels of anti-HS, anti-dsDNA, and anti-cardiolipin antibodies were detected in the sera of the immunized mice. An enzyme-linked immunospot (ELISPOT) assay indicted that IgG anti-HS and anti-dsDNA antibody-secreting cells were present in the kidneys and most likely contributed to antibody localization. Antibodies eluted from the kidneys of immunized mice were found to react strongly with HS and dsDNA when tested in vitro. The HS-immunized mice developed moderate to severe levels of proteinuria. Histologic examination of kidneys from HS-immunized mice revealed deposition of immunoglobulin in the kidneys. Our results describe the induction of SLE-like disease in normal mice following immunization with HS. This experimental model may be useful for understanding the immunologic basis for autoimmunity to HS.

Neutrophil-mediated damage to vascular endothelium in the spontaneously hypertensive rat.

Lysis of aortic endothelial cells (EC) by neutrophils from spontaneously hypertensive rats (SHR) was investigated using a nonradioactive cytotoxicity assay. Interleukin-1-activated EC, but not unstimulated EC, were effective target cells for lysis by SHR neutrophils. Supernatants from activated neutrophil did not exert a cytotoxic effect on EC. Inhibitors of reactive oxygen species did not affect the cytotoxicity of neutrophils on EC. In contrast, inhibitors of serine protease and elastase markedly inhibited the cytotoxicity of neutrophils on EC. Antibodies against the endothelial cell surface ligands ICAM-1 (CD54) and E-selectin (CD62E) inhibited the adhesion and cytotoxicity of activated neutrophils on EC. The cytotoxicity of neutrophils required direct cell-to-cell contact because separating them with a microporous membrane abrogated the neutrophil-mediated cytotoxic activity. These results demonstrate that SHR neutrophils possess potent cytotoxicity against cytokine-activated EC. Neutrophil-mediated damage of EC could contribute to organ damage in hypertension under conditions of local or systemic activation of neutrophils.

Characterization of arterial antigens using arterial antigen-reactive T cell clones from spontaneously hypertensive rats.

We have previously demonstrated that arterial antigens derived from the aorta of spontaneously hypertensive rats (SHRs) stimulate arterial antigen-reactive T cell clones established from the spleens of SHR to proliferate and release cytokines. To identify immunogenic protein components associated with the arterial wall, arterial antigen-reactive T cell clones were tested against arterial antigens separated by SDS-PAGE and transferred to nitrocellulose. The greatest T cell reactivity was obtained with protein bands of molecular weight 66 kDa, 50 kDa, and 45 kDa. T cell clones reactive against the 50 and 45 kDa antigens from gels failed to respond to proteins of other molecular weight (M(r)) separated under reducing or nonreducing conditions, suggesting that these molecules are not subunits of larger proteins and may represent monomeric antigens polymerized into the arterial wall. These data suggest that certain epitopes of arterial wall antigens are immunogenic. T cells activated with these immunogenic epitopes could initiate or perpetuate vasculitis in the arteries of hypertensive rats.

Isolation and functional characterization of IL-2 responsive T cell clones from NZB x NZW F1 mice.

Autoantigen-reactive T cells might play an important role in the pathogenesis of systemic lupus erythematosus (SLE). Autoantigen-reactive T cell clones were generated from spleens of NZB x NZW F1 (BWF1) and normal control BALB/c mice with interleukin-2 (IL-2), a procedure that selects for in vivo activated antigen-reactive T cells. The antigen-specificity of the T cell clones was tested by using a panel of candidate autoantigens. The T cell clones from BWF1 mice but not those from BALB/c mice proliferated against heparan sulfate, the major glycosaminoglycan of glomerular basement membrane. None of the clones proliferated against dsDNA or cardiolipin. All the heparan sulfate-reactive T cell clones had the ability to selectively augment the production of IgG anti-dsDNA autoantibodies. When cultured with either heparan sulfate or Concanavalin A, the T cell clones produced high levels of IL-4 and IL-5 with no detectable IL-2 or IFN-gamma. In contrast, T cell clones derived from BALB/c mice augmented the production of total polyclonal IgG but not the production of anti-dsDNA antibodies. These studies indicate the existence of heparan sulfate-reactive T cells in BWF1 mice. Characterization of heparan sulfate-reactive T cells that could selectively augment anti-dsDNA production will permit the design of targeted and antigen-specific therapy.

Autoantibodies against arterial antigens: characterization by ELISA and immunoblot analysis in the spontaneously hypertensive rat.

The presence of autoantibodies directed against arterial antigens in serum samples from spontaneously hypertensive rats and related controls that included Wistar-Kyoto and Sprague-dawley rats were assayed by enzyme-linked immunosorbent assay and immunoblotting technique. Circulating immunoglobulin G antibodies reactive against arterial antigen, as measured by enzyme-linked immunosorbent assay, could be detected in serum samples of 26 of 30 spontaneously hypertensive rats (87%) and 8 of 30 (27%) Wistar kyoto rats. These antibodies (Abs) were not detectable either by enzyme-linked immunosorbent assay or immunoblotting in sera from Sprague-dawley rats. The arterial antigen-reactive antibody was antigen specific, because the binding reactivity was absorbed by arterial antigen but not by fibroblasts or peripheral blood mononuclear cells. Immunoglobulin G arterial antigen-reactive antibody was significantly higher in adult spontaneously hypertensive rats with established hypertension, compared with young prehypertensive rats or normotensive wistar kyoto rats. Immunoblotting of spontaneously hypertensive rats sera revealed reactivity of arterial antigen-reactive antibody against arterial antigen ranging in size from 20 to 97 kDa. Sera from Wistar kyoto rats recognized arterial antigen ranging in size from 40 to 90 kDa. A significant correlation (p < 0.004) was found between adult spontaneously hypertensive rats with established hypertension and the presence of arterial antigen-reactive antibody reactivity against arterial antigen of 20, 69 and 97 kDa. Antibody directed against a 20 kDa arterial antigen was detected in both young prehypertensive rats and adult rats with established hypertension but not in Wistar kyoto or Sprague-dawley rats. Antibodies directed against both 69 and 97 kDa arterial antigens were detected only in spontaneously hypertensive rats sera. These data show that the pattern of arterial antigen-reactive antibody reactivity in sera of hypertensive rats in heterogeneous, and suggest that arterial antigen-reactive antibody directed against few arterial antigens may be involved in the pathogenesis of hypertension in the spontaneously hypertensive rat.

Production of anti-acetylcholine receptor-alpha antibody in vitro by peripheral blood lymphocytes of patients with myasthenia gravis: role of immunoregulatory T cells and monocytes.

To study the role of T cells in T and B cell interaction resulting in production of antibody (Ab) to the alpha chain of acetylcholine receptor (anti-AChR-alpha Ab) in myasthenia gravis (MG), we cocultured peripheral blood-purified B and T cells of patients with MG and of control subjects with and without multiple sclerosis in the presence of AChR-alpha or pokeweed mitogen. Under these conditions, a high level of anti-AChR-alpha Ab was produced by cells of patients with MG but not of control subjects. Production of anti-AChR-alpha Ab by B cells was stimulated by autologous purified or cloned CD4+ T cells, whereas autologous CD8+ T cells had no effect. CD8+ T cells did not suppress anti-AChR-alpha Ab production when added to B cells cocultured with CD4+ T cell clones. Anti-AChR-alpha Ab production was inhibited by monoclonal antibodies against CD4 and class II major histocompatibility complex (MHC) antigens, indicating that these antigens are required for productive T-B cell interactions resulting in anti-AChR-alpha Ab synthesis. Anti-AChR-alpha Ab production by peripheral blood lymphocytes of patients with MG was significantly lower than that by their purified or cloned T cells cultured with B cells. Cell-mixing experiments indicated that anti-AChR-alpha Ab synthesis was inhibited by monocytes. The prostaglandin synthetase inhibitor, indomethacin, partially restored the suppressive effect of monocytes on anti-AChR-alpha Ab synthesis. These results indicate that induction of anti-AChR-alpha Ab production by CD4+ T cell clones requires CD4 and class II MHC antigens and is inhibited by suppressor macrophages and not by CD8+ T cells.

Isolation of T-cell clones with specificity for arterial antigen from spontaneously hypertensive rats.

OBJECTIVE: It has been postulated that hypertension in the spontaneously hypertensive rat (SHR) results from autoimmune damage to the SHR vasculature. The objective of this study was to isolate autoreactive T-cells specific for arterial antigens, and to characterize these cells. DESIGN: The presence of autoreactive T-cells in the SHR has not been studied previously. Lymphocytes were isolated from spleens obtained from SHR and Wistar-Kyoto (WKY) rats aged 4, 8, 12, 16, 20, 24 and 28 weeks. METHODS: Limiting dilution analysis was used to clone and to establish arterial antigen-reactive T-cell clones. The specificity of these clones was assessed by measuring lymphokine production and T-cell proliferation induced by arterial antigen and by irrelevant control antigens. RESULTS: All of the SHR, regardless of age, possessed arterial antigen-specific CD4+, major histocompatability complex class II-restricted T-cells. The responses of freshly isolated spleen cells to arterial antigen were weaker than the proliferative responses of interleukin-2-expanded T-cells to arterial antigen. The T-cell clones also produced interleukin-2, interleukin-4 and interferon-gamma in response to arterial antigen. However, the presence of T-cells specific for arterial antigen is not unique to SHR, since a similar response was seen in normotensive WKY rats. CONCLUSIONS: The results indicate the existence of T-cells specific for arterial antigen in the spleens of both SHR and WKY rats. Thus, arterial antigen-reactive T-cells cannot be the initial cause of hypertension, but the activation of such autoreactive T-cells might be important in the development of hypertension.

The effects of PEG-interleukin-2 and interleukin-2 on essential hypertension and cellular immune function in the spontaneously hypertensive rat.

The effects of recombinant human interleukin-2 covalently linked to polyethylene glycol (PEG-IL-2) or interleukin-2 (IL-2) on hypertension and in vitro suppressor T cell function in the spontaneously hypertensive rats (SHR) were investigated. Male young prehypertensive (4 weeks old) SHRs and adult (10 weeks old) SHRs with established hypertension were injected with low (5,000 units (u)/kg) or high (50,000-100,000 u/kg) dose of PEG-IL-2 or IL-2 as a single bolus or repeated injections. Systolic blood pressure was measured twice weekly using the tail-cuff technique. Systolic blood pressure in the PEG-IL-2 or IL-2 treated animals, irrespective of age, dose, or route of injection, did not differ significantly from that measured in vehicle-treated controls over a 10 week period. Mean arterial pressure measured by intra-arterial catheter was 159 +/- 7 mm Hg 10 weeks after treatment with repeated injections of 5,000 u/kg of PEG-IL-2 and 158 +/- 9 mm Hg in vehicle-treated controls. All rats injected with IL-2 had IL-2-specific IgG antibody in their sera. None of the PEG-IL-2 treated rats had any detectable anti-IL-2 antibodies in their sera. Thus, PEG-IL-2 showed far less immunogenicity than IL-2. Suppressor T (Ts) cells generated from adult SHR spleen cells failed to suppress pokeweed mitogen (PWM)-driven immunoglobulin G (IgG) synthesis. PEG-IL-2 or IL-2 supplementation both in vitro and in vivo restored the ability of adult SHR to generate Ts cells able to inhibit IgG synthesis. Our data suggest that PEG-IL-2 or IL-2 administration does correct a prominent defective Ts cell activity found in adult SHR, but that correction of this immune abnormality is not attended by an attenuation of hypertension.

Elevated serum copper is associated with reduced immune response in aging mice.

Young mice were found to have serum copper concentrations ranging from a low of 0.291 to a high of 0.584 ppm. Old mice had serum copper concentrations ranging from 0.223 to 1.715 with 30.9% of the old animals having values greater than 0.6 ppm. The mitogen response of isolated lymphocytes from the spleens of aging mice was greatly reduced when these cells were taken from animals with naturally occurring serum copper levels in excess of 0.6 ng of copper/mg wet weight serum. The lymphocytes taken from young mice with higher serum copper concentrations, on the other hand, had increased response to mitogens. Addition of the copper protein, ceruloplasmin, to lymphocyte cultures in vitro reduced the mitogen response of purified splenic lymphocytes with the reduction being greater for cells from old animals. We suggest that excess serum copper and ceruloplasmin may be immunosuppressive, especially in older organisms.

Abnormal activation and loss of suppressor T cells in the spontaneously hypertensive rat.

Suppressor T cell function in the spontaneously hypertensive rat (SHR) and normotensive Wistar Kyoto (WKY) rats was analyzed using syngeneic mixed lymphocyte reaction (SMLR) and concanavalin A (Con A) activation. A depressed SMLR was found in adult SHR but not in adult WKY. IL-2 synthesized by SHR was 40-fold lower than that of WKY, and the suppressor T cells generated in the SMLR were incapable of suppressing IgG synthesis. Precursors of cells that can be activated by Con A to become functional suppressor cells are reduced in adult SHR. Supernatant fluids derived from Con A-activated spleen cells from adult SHR failed to significantly inhibit IgG synthesis by cultures of syngeneic spleen cells compared to supernatant fluids from young SHR or WKY Con A-activated spleen cells. However, spleen cells from both adult SHR and WKY proliferated strongly and released equivalent amounts of IL-2 in response to Con A. Addition of exogenous IL-2 to the SMLR cultures in vitro restored the ability of SHR T cells to respond in the SMLR, with generation of cells capable of suppressing IgG synthesis. Administration of SHR with IL-2 in vivo also restored the suppressor T cell function in the SMLR. These results suggest a defective suppressor T cell activation and loss of suppressor T cell activity as the SHR age.

Characterization of a T suppressor cell line that downgrades experimental allergic encephalomyelitis in mice.

A T-suppressor (Ts) cell line of CD8 phenotype was isolated from spleens of SJL/J mice that had recovered from experimental allergic encephalomyelitis (EAE) induced by injection of MBP-activated T cells. The Ts cell line inhibited the proliferation of MBP-sensitized T cells in vitro. Addition of recombinant IL-2 enhanced the Ts-mediated suppression. Adoptively transferred Ts line was able to downgrade EAE in mice subsequently challenged with MBP-activated T cells. The mechanism of suppression appeared to involve neither direct cytolysis of the effector T cells nor the production of a soluble suppressor factor. The findings suggest an in vivo role for suppressor T cells in the regulation of EAE.

Characterization of in vivo-activated T cell clones from peripheral blood of multiple sclerosis patients.

In vivo-activated interleukin-2 responsive T cell clones were generated from peripheral blood (PBL) of multiple sclerosis patients (MS) and normal subjects (N) by limiting dilution analysis. The frequency with which interleukin-2 responsive cells were cloned from PBL was higher in MS than N. CD8 was the predominant phenotype expressed by both MS (85%) and N (89%) clones. Seven clones from four MS patients but none from five N subjects specifically proliferated against myelin basic protein. These studies demonstrate the existence of MBP-reactive T cells in PBL of MS patients.

Phenotypic and functional characterization of T cells from patients with myasthenia gravis.

A study of cell surface phenotypes of PBL of myasthenia gravis (MG) patients showed that their T cells had a significantly higher percentage of 4B4+ T cells (the helper/inducer subset) than age- and sex-matched controls. The PBL of MG patients proliferated significantly higher than those of normal subjects (NS) in response to the purified alpha chain of the acetylcholine receptor (AChR). Anti-AChR antibody was present in sera of 88% of MG and none of the NS. The PBL B cells from MG only, when cultured with autologous T cells and stimulated with either pokeweed mitogen (69%), or AChR-alpha chain (38%), secreted antibody to AChR-alpha chain, whereas T and B cells alone secreted no antibody. T cells from PBL of MG patients were more readily cloned than T cells of NS, by limiting dilution, in the presence of recombinant IL-2 and in the absence of AChR-alpha chain. About 50% of T cell clones from MG patients, compared to none from NS, proliferated to AChR-alpha chain. This response was HLA-DR restricted. MG T cell clones did not display significant cytotoxic activity, as compared to control T cell clones. Our results indicate that in MG, 4B4+ regulatory T cells play their role in the pathogenesis of MG, not by cytotoxicity, but more likely by their ability to stimulate specific antibody production by B cells.

Reversible interleukin-2 response defects in systemic lupus erythematosus.

Limiting dilution analysis techniques were used to determine precursor frequencies for interleukin-2 (IL-2) responsive cells among the peripheral blood lymphocytes of patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis compared with healthy subjects. Response defects in SLE were found, but were of two types: reduced precursor frequencies with normal pattern of response (single-hit kinetics); and abnormal multi-hit responsiveness. These abnormalities were not more frequent statistically in those with active disease. Precursor frequencies of SLE peripheral blood lymphocytes were enhanced by resting the cells for up to 72 h prior to activation, and by adding exogenous IL-2 during the initial activation step. The IL-2 response defects of SLE are therefore reversible and may in part be secondary to other in vivo abnormalities, such as deficient IL-2 production.

Lymphocyte extracellular matrix interactions. Induction of interferon by connective tissue components.

Limiting dilution analysis was performed in the presence of interleukin 2 (IL-2) on lymphocytes isolated from the synovial fluid (SF) and peripheral blood (PB) of patients with rheumatoid arthritis (RA) and PB of normal donors. Clones of these 'spontaneously' IL-2-responsive cells from PB and SF were compared for their reactivity with components of the extracellular matrix (i.e. native or denatured type I or type II collagen and proteoglycan). It was determined that all clones from both PB and SF were activated to produce interferon (IFN) in the presence of any of the connective tissue components (CTC). Clones derived from normal PB behaved in a similar fashion but produced lower IFN-gamma levels. There was a synergy between the CTC and serum or plasma fibronectin, which was more apparent when soluble CTC were used as the stimuli rather than immobilized CTC. The fibronectin alone was unable to induce IFN-gamma production under any of the conditions tested (i.e. soluble or immobilized). These results demonstrate that clones of IL-2-responsive T cells can be activated by interactions with connective tissue components to produce IFN-gamma.

Interleukin 2 responsive T cell clones from rheumatoid and normal subjects: proliferative responses to connective tissue elements.

In vivo-activated interleukin 2 responsive T cell clones were generated from peripheral blood (PB) and synovial fluid (SF) of rheumatoid arthritis (RA) patients and from normal control PB. The specificity of these clones was assessed by measuring proliferation induced by the connective tissue elements (CTE) collagen types I and II, native and denatured, proteoglycans, and irrelevant control antigens. The cloned T cells from RA patients but not from normal subjects responded in vitro with proliferation to all CTE but not to control antigens purified protein derivative, ovalbumin, or lysozyme. Proliferation occurred in the presence and absence of accessory cells (AC), but the responses were consistently higher in the presence of AC. Antibodies to HLA-DR abrogated the proliferative response to CTE suggesting that DR expression was necessary for the induction of proliferation. These findings demonstrate the existence of clonable T cells responsive to CTE in PB and SF of RA patients. Expression of reactivity to CTE may contribute to the chronicity of the inflammation in RA.

Fibroblast-activating factor production by interleukin (IL)-2 dependent T-cell clones from rheumatoid arthritis patients and normal donors.

T cells spontaneously responsive to interleukin (IL)-2 were cloned from the peripheral blood (PBL) or synovial fluid (SFL) lymphocyte populations obtained from patients with rheumatoid arthritis (RA) or from normal PBL. The clones were compared for their capacities to produce fibroblast-activating factors (FAFs) which support the growth of RA synovial fibroblasts. Clones derived from all sources produced FAFs following mitogen stimulation, but SFL clones synthesized significantly higher levels of FAF activity. Physicochemical characterization of the FAFs suggested that SFL- and PBL-derived clones produced similar factors. It was also demonstrated that interferon-gamma and IL-2 did not contribute to the FAF activity of the clone supernatants. These results demonstrate that a subpopulation of activated lymphocytes which are present in increased numbers in the rheumatoid joint can produce factors which influence rheumatoid synovial cell growth.

Characterization of IL-2 responsive synovial T lymphocytes in rheumatoid arthritis. II. Functional properties.

Lymphocytes from peripheral blood (PBL) and synovial fluid (SFL) were obtained from patients with rheumatoid arthritis (RA) and cloned under limiting-dilution conditions without prior activation but in the presence of exogenous interleukin (IL)-2. The precursor frequencies of such in vivo activated IL-2-responsive cells were higher in RA SFL (1/83) than in RA PBL (1/201) or normal PBL (1/377). These HLA-Dr/Ia-positive clones expressed T-cell markers CD3 and T101 and were either CD4- or CD8-positive but lacked NK markers CD11, CD16, and HNK-1. All such clones were cytotoxic for NK-sensitive K562 targets and NK-insensitive Raji cell targets. These cells, which most closely resemble nonmajor histocompatibility complex (MHC) restricted cytotoxic T (CTL) cells, are present with increased frequency in RA synovial fluids.

Characterization of synovial T lymphocytes in rheumatoid arthritis. I. Production of IL-2 dependent T cell clones from synovial fluid and peripheral blood.

Lymphocytes obtained from the peripheral blood (PBL) or synovial fluids (SFL) of patients with rheumatoid arthritis (RA) or other inflammatory joint diseases were compared with the PBL from normal individuals, by cloning under limiting dilution conditions in the presence of interleukin 2 (IL-2). The precursor frequency estimates of IL-2 responsive cells from these sources did not differ appreciably. However there were marked differences in the surface marker phenotypes of the clones derived from the PBL as compared to SFL. There was a predominance of OKT4-8+ cells in SFL from RA and non RA donors with inflammatory joint disease while PBL from all sources showed a marked prevalence of OKT4+8- cells. Comparison of precursor frequencies in the presence of PBL and SFL indicated that there were variations in the capacities of the SFL and PBL IL-2 dependent cells to grow on these fillers. SFL derived cells grew equally well on PBL or SFL filler, while PBL clones grew efficiently only on PBL fillers. Collectively these results indicate that there are marked differences in the surface phenotypes and growth requirements of IL-2 responsive SFL as compared to PBL.