Vicki E. Kelley

The Effect of Dietary Supplementation with n—3 Polyunsaturated Fatty Acids on the Synthesis of Interleukin-1 and Tumor Necrosis Factor by Mononuclear Cells

We examined whether the synthesis of interleukin-1 or tumor necrosis factor, two cytokines with potent inflammatory activities, is influenced by dietary supplementation with n-3 fatty acids. Nine healthy volunteers added 18 g of fish-oil concentrate per day to their normal Western diet for six weeks. We used a radioimmunoassay to measure interleukin-1 (IL-1 beta and IL-1 alpha) and tumor necrosis factor produced in vitro by stimulated peripheral-blood mononuclear cells. With endotoxin as a stimulus, the synthesis of IL-1 beta was suppressed from 7.4 +/- 0.9 ng per milliliter at base line to 4.2 +/- 0.5 ng per milliliter after six weeks of supplementation (43 percent decrease; P = 0.048). Ten weeks after the end of n-3 supplementation, we observed a further decrease to 2.9 +/- 0.5 ng per milliliter (61 percent decrease; P = 0.005). The production of IL-1 alpha and tumor necrosis factor responded in a similar manner. Twenty weeks after the end of supplementation, the production of IL-1 beta, IL-1 alpha, and tumor necrosis factor had returned to the presupplement level. The decreased production of interleukin-1 and tumor necrosis factor was accompanied by a decreased ratio of arachidonic acid to eicosapentaenoic acid in the membrane phospholipids of mononuclear cells. We conclude that the synthesis of IL-1 beta, IL-1 alpha, and tumor necrosis factor can be suppressed by dietary supplementation with long-chain n-3 fatty acids. The reported antiinflammatory effect of these n-3 fatty acids may be mediated in part by their inhibitory effect on the production of interleukin-1 and tumor necrosis factor.

Interaction of mutant lpr gene with background strain influences renal disease.

The mutant gene lpr on the MRL/Mp strain of mice is responsible for converting a late onset glomerulonephritis into an early, aggressive, and fatal renal disease. This gene induces the proliferation of a unique subset of lymphocytes, the production of a variety of autoantibodies and shortened survival in MRL/Mp as well as in the genetically distinct strains C3H/HeJ, C57BL/6J, and AKR/J. The present study examined in detail the role of the lpr gene in the formation of lupus nephritis. The results show that C3H-lpr and B6-lpr mice do not develop nephritis while the AKR-lpr strain has a mild form of renal disease. None of these newly constructed congenic mutant strains have the severity of proteinuria or the degree of renal pathology characteristic of MRL-lpr mice. Thus, the lpr gene alone is insufficient in producing severe renal injury. The interaction of the lpr gene with other factors is required for the induction of life-threatening lupus nephritis.

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 cyclooxy-genase 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<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<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 relanted 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 adids suppressed the increase in TxA2 and decreased the vasodilators PGE2 and PGI2 in the kidney and peritoneal macrophages, while modifying the glomerular filtration rate and vacuolization in proximal convoluted tubules characteristic of rodent CsA-associated nephrotoxicity. Thus, CsA-initiated renal vasoconstriction in related to an increase 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.

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, hetero topic 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.

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 CsAs 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 × 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.

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.

Effect of Ibuprofen, a fatty acid cyclooxygenase inhibitor, on murine lupus

Abstract Pharmacologic doses on prostaglandin E or autoimmune NZB × W mice inhibits the development of lupus. In the present study we evaluated the relationship of endogenous PGE production on the development of lupus by inhibiting the cyclooxygenase pathway with Ibuprofen. Daily injections of this agent did not modify survival, the development of proteinuria, or circulating amounts of antibodies to double-and single-stranded DNA or retroviral gp70 immune complexes. Although the site of immune complex localization in the glomerular basement membranes of treated mice was partially shifted from subepithelial to subendothelial position, renal disease developed at the same rate. Thus, chronic inhibition of products of the cyclooxygenase pathway alone in the NZB × W mice does not appear to mediate murine lupus.

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.

Interleukin 2 Toxin: A Step Toward Selective Immunomodulation

We have used protein engineering and recombinant DNA methodologies to genetically replace the eukaryotic cell receptor binding domain of diphtheria toxin with interleukin 2 (IL-2). The toxin-related T cell growth factor fusion gene has been cloned in Escherichia coli K12. Recombinant strains of E coli produce a 68,086 K hybrid toxin, IL-2 toxin that retains immunologic properties intrinsic to both its diphtheria toxin and IL-2 components. IL-2 toxin has been found to selectively inhibit protein synthesis in both human and murine T cell lines that bear high affinity IL-2 receptors, whereas the hybrid toxin is not active against cells that do not bear this receptor. The cytotoxic action of IL-2 toxin is specifically blocked by free IL-2 and monoclonal antibodies that bind to the p55 (Tac antigen) subunit of the high affinity IL-2 receptor. In addition, IL-2 toxin, like diphtheria toxin itself, must pass through an acidic compartment in order to deliver its adenosine diphosphate ribosyl transferase activity to the cytosol of target T cells. In a murine delayed type hypersensitivity (DTH) model system, we have shown that IL-2 toxin treatment induces a marked immunosuppression.