Selective suppression of retroviral gp70-anti-gp70 immune complex formation by prostaglandin E1 in murine systemic lupus erythematosus.

The effect of pharmacologic quantities of prostaglandin E1 (PGE) was investigated in three strains of mice (NZB X NZW, MRL/1, and BXSB) that spontaneously develop lupus-like glomerulonephritis. PGE-treatment prolonged survival and retarded the glomerular deposition of immune complex (IC) and the development of glomerulonephritis in NZB X NZW and MRL/1 mice, but did not similarly protect BXSB mice. Changes in the responsive strains correlated well with reduced amounts of circulating gp70 complexed with anti-gp70 antibodies compared with untreated controls, although total concentrations of gp70 (free and complexed) detectable in sera were similar in both groups of mice. The results strongly suggest that: (a) PGE selectively suppressed the immune response to retroviral gp70, (b) PGe had little effect on the quantity or quality of anti-DNA antibodies but did reduce the deposition of anti-DNA containing IC in the kidneys, and (c) gp70 IC appear to play an important role in the pathogenesis of glomerulonephritis in murine systemic lupus erythematosus.

Administration of an anti-interleukin 2 receptor monoclonal antibody prolongs cardiac allograft survival in mice.

Administration of the monoclonal antibody M7/20, which binds to the murine interleukin-2 (IL) receptor, significantly prolongs cardiac allograft survival in two H-2-incompatible strain combinations of inbred mice. The results support the important role of the IL-2 receptor in the mechanism of graft rejection, and suggest its suitability as a target for immunosuppressive therapy.

Increased renal thromboxane production in murine lupus nephritis.

To determine whether the amount of cyclooxygenase metabolites correlates with the development of lupus nephritis, intrarenal eicosanoid production was measured in autoimmune mice. Disease progression was related to the renal biosynthesis of prostaglandin (PGE2), prostacyclin (6 keto PGF1 alpha), and thromboxane (TXB2) using the MRL-lpr and NZB X NZW F1 hybrid mouse strains with predictably progressive forms of renal disease that mimic the human illness. Mice were evaluated for renal disease by measuring urinary protein excretion and renal immunopathological conditions and these features were related to renal eicosanoid production. These studies show that: (a) intrarenal synthesis of TXB2 increased incrementally in MRL-lpr and NZB X NZW F1 hybrid mice as renal function deteriorated and renal pathologic events progressed; (b) there were no consistent increases in the levels of two other cyclooxygenase metabolites, PGE2 or 6 keto PGF1 alpha; (c) increased TXB2 production occurred in the renal medulla, cortex, and within enriched preparations of cortical glomeruli; (d) when renal disease was prevented by pharmacologic doses of PGE2, intrarenal TXB2 did not increase; (e) administration of a dose of ibuprofen (9 mg/kg), a cyclooxygenase inhibitor capable of reducing 90% of platelet TXB2 without affecting intrarenal levels, did not retard the progression of renal damage. Taken together, these data indicate that the intrarenal level of TXB2 rises in relation to the severity of murine lupus nephritis. Furthermore, because of the potential deleterious effects of TXA2, enhanced production of this eicosanoid may be an important mediator of renal injury.

Decreased expression of human class II antigens on monocytes from patients with acquired immune deficiency syndrome. Increased expression with interferon-gamma.

The expression of HLA-DR (a class II histocompatibility antigen) on monocytes isolated from the peripheral blood of normal individuals and patients with acquired immune deficiency syndrome (AIDS) was investigated by the use of dual fluorescent staining and cytofluorometry. In animal models the absence of class II positive monocytes is linked to a failure of T cells to respond to antigens. We now report that patients with AIDS have a paucity of HLA-DR+ monocytes. The percentage of HLA-DR+ monocytes among eight normal individuals ranged from 49.3 to 95.0%+, and only one individual had less than 50% HLA-DR+ monocytes. HLA-DR expression on monocytes from homosexual male patients with lymphadenopathy was similar to that of normal subjects (range, 58.0 to 97.4%+). In contrast, seven of nine patients with AIDS had less than 50% HLA-DR+ monocytes (range, 13.4 to 78.8%+). The in vitro incubation of monocytes from AIDS patients with cloned human interferon-gamma resulted in an increase of the expression of HLA-DR to near normal levels.

Use of nuclear antigen-coated red cells in hemolytic plaque assays.

By studying antinuclear antibody production at the cellular level, we can better understand the problems of immunoregulation in individuals with systemic lupus erythematosus. To date, the use of hemolytic plaque assays to detect B cells secreting antinuclear antibodies has been hampered by an inability to achieve reliable coating of red cells by nuclear antigens. Because the chromic chloride technique has proved ineffective for coupling nucleic acids and/or nuclear antigens to sheep red blood cells (SRBC) in our laboratory, we have developed a method of coupling SS DNA, DS DNA, poly(I).poly(C), Sm, and ENA to red cells pretreated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (ECDI) and poly(L-lysine) (PLL). Coated red cells were agglutinated by specific antisera and not by normal sera and were then used in hemolytic plaque assays to detect antinuclear plaque-forming cells (PFC) in spleens from various strains of mice with lupus-like syndromes. PFC specific for SS DNA, DS DNA, poly(I).poly(C), Sm, and ENA were found in MRL/lpr and NZB x W mice, and the number of anti-SS DNA and anti-DS DNA PFC correlated with the age of the animals. Indirect (IgG) PFC specific for nuclear antigens increased dramatically in female NZB x W mice between 11 and 13 months, a time when more than 50% of the animals usually die. Preliminary studies have shown that PFC specific for nuclear antigens can be detected in peripheral blood from patients with lupus erythematosus. Pretreatment of sheep red cells with ECDI and PLL thus allowed the coupling of selected nuclear antigens to these cells and provided the first demonstration of IgM and IgG PFC specific for a variety of nuclear antigens.

Modification of experimental nephrotoxicity with fish oil as the vehicle for cyclosporine.

Cyclosporine-associated renal dysfunction is well recognized. While renal vasoconstriction appears to be a major pathogenic factor, the precise mechanism responsible for the altered hemodynamics is unclear. To investigate whether alterations in renal eicosanoid metabolism could be involved, we substituted fish oil rich in eicosapentaenoic acid (EPA), an inhibitor of cyclooxygenase metabolites, for the conventional olive oil cyclosporine vehicle. Male rats were pretreated with 1.0 cc fish oil or olive oil by gavage. After 14 days, cyclosporine (12.5 mg/cc vehicle) was added to the oil and animals received cyclosporine 50 mg/kg for an additional 14 days. Pair-fed control animals received fish oil or olive oil alone. Glomerular filtration rate (GFR) was severely reduced in the cyclosporine-in-olive-oil (CSA + OO) group (0.28 +/- .05 ml/min/100 g) vs. olive oil (OO) controls (0.70 +/- .04) (P less than 0.001). While GFR was reduced in the cyclosporine-in-fish oil group (CSA + FO) vs. fish oil (FO) controls (0.47 +/- .07 vs. 0.74 +/- .04), it was significantly higher than in the CSA + OO group (P less than 0.05). Trough whole-blood cyclosporine levels were not significantly different in the two groups. While CSA + OO appeared to elevate renal cortical content of thromboxane B2 (65.7 +/- 7.3 pg/mg tissue vs. 46.9 +/- 5.3 for OO), both the CSA + FO and FO groups had reduced levels (31.1 +/- 2.7 and 29.5 +/- 2.3, respectively). In addition, there was a striking reduction in proximal tubular vacuolar changes in the CSA +/- FO vs. CSA + OO group. We conclude that the use of EPA-rich fish oil as the vehicle for cyclosporine results in improved renal function and morphology and is associated with depressed renal cortical levels of vasoconstrictor thromboxane B2.

Selective enhancement of thromboxane in macrophages and kidneys in cyclosporine-induced nephrotoxicity. Dietary protection by fish oil.

Cyclosporine (CsA) associated renal dysfunction is related in part to renal vasoconstriction. To identify the role of cyclooxygenase metabolites in the induction of vasoconstriction, we analyzed the effect of CsA on the synthesis of thromboxane (TxA2) prostacyclin (PGI2) and prostaglandin E2 (PGE2) in the kidney and peritoneal macrophages. Groups of rats were pair-fed diets enriched with 20% fish oil (FO) or corn oil (CO) for 4 weeks and then were injected with CsA 12.5 mg/kg/day i.p. for 2 weeks. CsA induced the synthesis of TxA2 and modestly reduced PGE2 and PGI2 in renal cortex and peritoneal macrophages. Feeding rats a diet enriched in FO containing omega-3 fatty acids as compared with CO without these fatty acids suppressed the increase in TxA2 and decreased the vasodilators PGE2 and PGI2 in the kidney and peritoneal macrophages, while modifying the decrease in the glomerular filtration rate and vacuolization in proximal convoluted tubules characteristic of rodent CsA-associated nephrotoxicity. Thus, CsA-initiated renal vasoconstriction is related to an increase in the vasoconstrictive Tx molecule and the reduction in vasodilator metabolites. Intrarenal macrophages represent a likely source of this Tx production. Feeding rats diets containing omega-3 fatty acids, known to be competitive inhibitors of cyclooxygenase metabolites, prevents CsA from selectively increasing TxA2 and preserves renal function.

Interleukin-2-diphtheria toxin fusion protein prolongs murine islet cell engraftment.

Allograft rejection is dependent upon complex cell-mediated processes, the primary effectors of which are activated T cells. As the expression of the cell surface protein interleukin 2 receptor is primarily limited to the subset of stimulated T cells, therapeutic agents that target this molecule may provide highly selective immunosuppression. A newly constructed chimeric IL-2 diphtheria toxin fusion protein specifically binds to and poisons activated T cells bearing the high-affinity IL-2R. We describe the in vivo effects of IL-2 toxin in preventing rejection of a crude pancreatic islet preparation transplanted across major and minor histoincompatibility barriers. IL-2 toxin administered once daily as the sole immunosuppressive agent prolongs islet graft survival and decreases the severity of the early mononuclear cell infiltrate into the graft site. Long-term survival of transplanted islets (greater than 100 days) was achieved following a short course (10 days) of more-intensive IL-2 toxin treatment. Thus IL-2 toxin, a highly selective immunosuppressive agent, leads to prolonged islet cell engraftment while sparing the resting or memory subset of the entire T cell repertoire.

Enhancement of immunosuppression by substitution of fish oil for olive oil as a vehicle for cyclosporine.

As previously reported, acute cyclosporine-induced nephrotoxicity is characterized by a decline in glomerular filtration rate and a selective intrarenal production of the vasoconstrictor thromboxane (TxA2), but not vasodilator prostaglandin E2 (PGE2), or prostacyclin (PGI2), cyclooxygenase metabolites. Fish oils (FO), that are rich in n-3 polyunsaturated fatty acids have a high affinity for cyclooxygenase but serve as poor substrate inhibit TxA2 synthesis. We have shown that when FO replaces olive oil (OO) as the vehicle for CsA, CsA-induced nephrotoxicity and increased TxA2 synthesis are obviated in rodent models. In this study, we demonstrate that the FO vehicle for CsA does not compromise CsA's immunosuppressive properties as deduced from studies of a delayed-type hypersensitivity (DTH) model in BALB/c mice and in a rat heart transplant model. In fact, concurrent FO administration with CsA actually enhances immunosuppression. A dose of CsA incapable of blunting DTH when injected in OO was suppressive when given in FO. Administration of as little as 0.05 ml of FO vehicle potentiated the suppressive action of CsA. In addition, nonconcurrent dietary supplementation of FO in animals receiving CsA caused an increase in the immunosuppressive action of CsA in DTH. FO alone reduced DTH as compared with OO, but was far less effective than CsA plus FO. Furthermore, doses of CsA (5 mg/kg/day or 1.5 mg/kg/day), which are subtherapeutic when administered with OO, prolonged engraftment of Lewis recipients of Lewis x Brown-Norway F1 hearts when CsA was solubilized with FO. These studies indicate that concurrent administration of CsA and FO potentiates the activity of CsA and thus increases its therapeutic index. Thus, CsA plus FO is potentially a safe, potent antirejection therapy worthy of clinical testing, especially insofar as FO prevents CsA-induced acute nephrotoxicity in the rodent.

Dose-related mechanisms of immunosuppression mediated by murine anti-CD3 monoclonal antibody in pancreatic islet cell transplantation and delayed-type hypersensitivity.

Although anti-CD3 mAb therapy is used extensively in clinical transplantation, the dose-related effects and mechanisms of action are not clearly defined. We have examined the dose-related effects of an antimurine CD3 mAb, 145-2C11, in pancreatic islet cell allograft and the delayed type hypersensitivity reaction models of T-cell-dependent immunity. Low-dose anti-CD3 therapy (0.5 micrograms/day) administered over several days mediated superficially equal, effective clinical immunosuppression as a single high-dose intravenous injection (400 micrograms). T cells harvested from animals treated with high-dose anti-CD3 were unresponsive to in vitro restimulation. In contrast, T cells isolated from low-dose treated animals retained in vitro proliferative capacity when restimulated with polyvalent anti-CD3 mAb. The terminal complement components were not required to support in vivo immunosuppression mediated by anti-CD3 mAb as C5 deficient mice were immunosuppressed by the administration of this mAb. In some pancreatic islet cell allograft recipients, permanent engraftment, but not tolerance, was achieved. Replacement of donor leukocytes produced acute rejection in hosts bearing long-term, well-accepted grafts. Prolonged anti-CD3 mAb treatment may provide sufficient time for replacement or inactivation of donor leukocytes.

The effect of anti-interleukin-2 receptor monoclonal antibody on allograft rejection.

During immune response to an allograft, activated T cells express a number of cell surface activation antigens, among them the membrane receptor for the lymphokine interleukin 2 (IL-2). As the IL-2 receptor is not present on resting T cells, it offers an attractive target for potentially specific immunosuppressive therapy. The rat monoclonal antibody M7/20, which binds to the murine IL-2 receptor, was studied for its effect on allograft survival in two H-2-incompatible strain combinations in inbred mice. Treatment with M7/20 for 10 days markedly prolonged survival of vascularized, heterotopic heart allografts in both strain combinations, with indefinite graft survival in 50% of recipients. The same treatment significantly prolonged skin allograft survival in one of the two combinations. The results support the important role of the IL-2 receptor in the mechanism of graft rejection and confirm its suitability as a target for immunosuppressive therapy in transplantation.

Immunotoxins and cytokine toxin fusion proteins.

Paul Ehrlich first suggested the simple and elegant concept of creating specific cell toxins or "magic bullets" through the fusion of cell-specific antibodies and toxins. In practice it has proven difficult to create safe and effective "magic bullets." In the past several years, several immunotoxins have been applied to clinical testing. These immunotoxins have been created by the biochemical coupling of cell- or lineage-specific monoclonal antibodies to plant toxins or fragments thereof. These immunotoxins have been used to treat bone marrow transplant recipients and patients with autoimmune disorders. In recent years, another strategy has also been pursued to create hybrid toxins. Rather than use antibodies as the targeting moiety, cytokines have been used to target a select population of cells bearing a high copy number of receptors for the specific cytokine. Rather than biochemically couple a cytokine to the toxin, the cytokine and toxin are fused by a peptide bond established via genetic engineering. A prototype IL-2 diphtheria toxin-related fusion protein is now being tested in the clinic for treatment of hematopoietic malignancies and autoimmune disorders.