Peter I. Lobo

NKT Cell Activation Mediates Neutrophil IFN-γ Production and Renal Ischemia-Reperfusion Injury

Previous work has shown that ischemia-reperfusion (IR) injury (IRI) is dependent on CD4+ T cells from naive mice acting within 24 h. We hypothesize that NKT cells are key participants in the early innate response in IRI. Kidneys from C57BL/6 mice were subjected to IRI (0.5, 1, 3, and 24 h of reperfusion). After 30 min of reperfusion, we observed a significant increase in CD4+ cells (145% of control) from single-cell kidney suspensions as measured by flow cytometry. A significant fraction of CD4+ T cells expressed the activation marker, CD69+, and adhesion molecule, LFA-1high. Three hours after reperfusion, kidney IFN-γ-producing cells were comprised largely of GR-1+CD11b+ neutrophils, but also contained CD1d-restricted NKT cells. Kidney IRI in mice administered Abs to block CD1d, or deplete NKT cells or in mice deficient of NKT cells (Jα18−/−), was markedly attenuated. These effects were associated with a significant decrease in renal infiltration and, in activation of NKT cells, and a decrease in IFN-γ-producing neutrophils. The results support the essential role of NKT cells and neutrophils in the innate immune response of renal IRI by mediating neutrophil infiltration and production of IFN-γ.

The chemokine receptors CCR2 and CX3CR1 mediate monocyte/macrophage trafficking in kidney ischemia–reperfusion injury

Chemokines and their receptors such as CCR2 and CX3CR1 mediate leukocyte adhesion and migration into injured tissue. To further define mechanisms of monocyte trafficking during kidney injury we identified two groups of F4/80-positive cells (F4/80(low) and F4/80(high)) in the normal mouse kidney that phenotypically correspond to macrophages and dendritic cells, respectively. Following ischemia and 3 h of reperfusion, there was a large influx of F4/80(low) inflamed monocytes, but not dendritic cells, into the kidney. These monocytes produced TNF-alpha, IL-6, IL-1alpha and IL-12. Ischemic injury induced in CCR2(-/-) mice or in CCR2(+/+) mice, made chimeric with CCR2(-/-) bone marrow, resulted in lower plasma creatinine levels and their kidneys had fewer infiltrated F4/80(low) macrophages compared to control mice. CX3CR1 expression contributed to monocyte recruitment into inflamed kidneys, as ischemic injury in CX3CR1(-/-) mice was reduced, with fewer F4/80(low) macrophages than controls. Monocytes transferred from CCR2(+/+) or CX3CR1(+/-) mice migrated into reperfused kidneys better than monocytes from either CCR2(-/-) or CX3CR1(-/-) mice. Adoptive transfer of monocytes from CCR2(+/+) mice, but not CCR2(-/-) mice, reversed the protective effect in CCR2(-/-) mice following ischemia-reperfusion. Egress of CD11b(+)Ly6C(high) monocytes from blood into inflamed kidneys was CCR2- and CX3CR1-dependent. Our study shows that inflamed monocyte migration, through CCR2- and CX3CR1-dependent mechanisms, plays a critical role in kidney injury following ischemia reperfusion.

Racial disparities in renal transplant outcomes

The purpose of our study was to evaluate the association of race and ethnicity with outcomes in the living related donor (LRD) renal transplant population, using multivariable adjustment for potential confounding variables. We prospectively analyzed 14,617 patients from the UNOS Renal Transplant Registry who underwent LRD renal transplantations in the United States between January 1, 1988 and December 31, 1996 using the Cox proportional hazards model. This model adjusts for the effects of potential genetic, social, and demographic confounding variables that may be associated with race or ethnicity long-term graft survival. Blacks were 1.8 times as likely as whites (P < 0.01, RR = 1.77) to suffer graft failure during the 9-year study period, which decreased minimally to 1.7 (P < 0.01, RR = 1.65) after controlling for potential confounding variables. Neither genotypic nor phenotypic HLA matching improved outcomes in blacks. Black renal transplant recipients had lower graft survival even after adjustment for matching and rejection, suggesting that non-HLA or socioeconomic mechanisms may contribute to racial differences in transplantation outcomes.

Immunomodulatory effect of Nigella sativa proteins fractionated by ion exchange chromatography.

Whole Nigella sativa (N. sativa) proteins were purified on a DEAE Sephadex A50 ion exchange column. Complete fractionation was achieved in four peaks. Analysis of the purified peaks was carried out by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Whole N. sativa showed a number of protein bands ranging from 94-10 kDa molecular mass. In mixed lymphocyte cultures (MLC), whole N. sativa and its purified proteins were found stimulatory as well as suppressive and this effect varied from one donor to another. Maximum stimulation (mean + S.E. of % relative index was 63.73 + 20.78) was observed with fractionated N. sativa proteins (P1) (10 microg/ml) in MLC. In MLC, also N. sativa peaks (P1 and P2) were stimulatory at all concentrations (10 microg/ml, 1 microg/ml or 0.1 microg/ml) used. However, a uniformly suppressive effect of N. sativa and its all four peaks at a concentration of 10 microg/ml was noticed when lymphocytes were activated with pokeweed mitogen (PWM). The effect of N. sativa proteins was further evaluated on the production of cytokines which were measured by using specific enzyme-linked immunosorbent assay. Large quantities of IL-1beta were secreted by whole N. sativa in culture medium with non-activated peripheral blood mononuclear cells (PBMC) (450 pg/ml) and with allogeneic cells (410 pg/ml). Fractionated N. sativa was less effective when compared with whole N. sativa proteins. No effect on IL-4 secretion was seen either by using non-activated, PWM-activated or allogeneic-cells. Whole N. sativa suppressed as well as stimulated the production of IL-8 in non-activated and PWM-activated PBMC respectively. All N. sativa peaks with protein concentration of 2 microg/ml were stimulatory for the induction of IL-8 by PWM-activated cells. However, no effect on IL-8 was seen either with whole N. sativa or its peaks when allogeneic PBMC were used. Stimulatory effect of whole N. sativa and fractionated proteins was also noticed on the production of TNF-alpha either using non-activated or mitogen activated cells.

Activation of Adenosine 2A Receptors Attenuates Allograft Rejection and Alloantigen Recognition

The current studies investigated the in vitro and in vivo effect of adenosine 2A receptor (A2AR) agonists to attenuate allogenic immune activation. We performed MLRs with spleen T lymphocytes and APCs isolated from wild-type and A2AR knockout mice of both C57BL/6 and BALB/c background strains. Two-way MLR-stimulated T cell proliferation was reduced by ATL313, a selective A2AR agonist in a dose-responsive manner (∼70%; 10 nM), an effect reversed by the A2AR antagonist ZM241385 (100 nM). By one-way MLRs, we observed that ATL313’s inhibitory effect was due to effects on both T cells and APCs. ATL313 suppressed the activation markers CD25 and CD40L and the release of inflammatory cytokines IFN-γ, RANTES, IL-12P70, and IL-2. ATL313 also increased negative costimulatory molecules programmed death-1 and CTLA-4 expressed on T cells. In lymphocytes activated with anti-CD3e mAb, ATL313 inhibited the phosphorylation of Zap70, an effect that was reversed by the protein kinase A inhibitor H-89. In skin transplants, allograft survival was enhanced with ATL313, an effect blocked by ZM241385. These results indicate that A2AR agonists attenuate allogenic recognition by action on both T lymphocytes and APCs in vitro and delayed acute rejection in vivo. We conclude that A2AR agonists may represent a new class of compounds for induction therapy in organ transplantation.

Evidence demonstrating poor kidney graft survival when acute rejections are associated with IgG donor-specific lymphocytotoxin.

The current prospective investigation was conducted to determine whether development of IgG donor-specific lymphocytotoxins detected at the onset of acute rejections was predictive of a poor-prognosis acute rejection. Between January 1990 and August 1993, 206 kidney transplants were performed. Cadaver kidney recipients were managed with antilymphocyte globulin as induction therapy and all recipients (i.e., cadaver and living related donor kidneys) received triple immunosuppressive therapy, i.e., CsA, AZA, and prednisone. Rejections were treated with intravenous Solu-Medrol and OKT3. Presence of donor-specific IgG lymphocytotoxin was detected by using dithiothreitol-pretreated sera (obtained at onset of rejection) and frozen donor cells. In addition, percentage of panel reactive antibody was determined on this dithiothreitol-pretreated sera. Of the 82 patients with biopsy-proven acute rejections, 19 were found to have developed donor-specific IgG lymphocytotoxin and a marked increase in panel reactive antibody. One-year graft survival in this group was dismal (16%), despite OKT3 therapy. Over 90% of these patients lost their graft within 2 months of rejection diagnosis. In 63 recipients who had acute rejections without development of IgG anti-HLA antibody, 1-year graft survival was 72%. The majority of these patients lost their grafts from chronic rejection. No anti-HLA activity was found in patients who did not have rejection episodes. Based on this study, evidence indicates that assaying for IgG donor-specific antibody at time of rejection is a valuable tool for selecting a subset of patients with poor-prognosis acute rejections. Identifying this subset will become important as we enter an era of new immunosuppressive agents.

Nigella sativa: effect on human lymphocytes and polymorphonuclear leukocyte phagocytic activity.

The effects of Nigella sativa (N. sativa) seeds and their soluble fractions were studied in vitro on lymphocyte response to different mitogens and on polymorphonuclear leukocyte phagocytic activity. No stimulatory effect of N. sativa was detected on lymphocyte response to phytohemagglutinin, concanavalin-A or pokeweed mitogen. A stimulatory effect of N. sativa was noticed on the lymphocyte response to pooled allogeneic cells. This effect was more pronounced when the low molecular weight (< 10 kDa) fraction was used and varied from one normal individual to another (25% to 825%). N. sativa enhanced the production of interleukin-3 by human lymphocytes when cultured with pooled allogeneic cells or without any added stimulator. N. sativa did not, however, enhance or suppress interleukin-2 secretion by mitogen activated peripheral blood mononuclear cells. Interestingly, N. sativa increased interleukin-1 beta, suggesting therefore, that it has an effect on macrophages. It also suppressed the leukocyte chemiluminescence activity using phorbol myristate acetate and Zymosan as stimulants. No effect of N. sativa or its fractions was, however, noticed on bacterial phagocytosis or killing when Staphylococcus aureus was used, indicating that the decrease in chemiluminescence activity in the presence of N. sativa is not relevant to the bactericidal activity.

Dendritic cells tolerized with adenosine A2AR agonist attenuate acute kidney injury

DC-mediated NKT cell activation is critical in initiating the immune response following kidney ischemia/reperfusion injury (IRI), which mimics human acute kidney injury (AKI). Adenosine is an important antiinflammatory molecule in tissue inflammation, and adenosine 2A receptor (A₂AR) agonists protect kidneys from IRI through their actions on leukocytes. In this study, we showed that mice with A₂AR-deficient DCs are more susceptible to kidney IRI and are not protected from injury by A₂AR agonists. In addition, administration of DCs treated ex vivo with an A₂AR agonist protected the kidneys of WT mice from IRI by suppressing NKT production of IFN-γ and by regulating DC costimulatory molecules that are important for NKT cell activation. A₂AR agonists had no effect on DC antigen presentation or on Tregs. We conclude that ex vivo A₂AR-induced tolerized DCs suppress NKT cell activation in vivo and provide a unique and potent cell-based strategy to attenuate organ IRI.

Autocrine Adenosine Signaling Promotes Regulatory T Cell–Mediated Renal Protection

Regulatory T cells (Tregs) suppress the innate inflammation associated with kidney ischemia-reperfusion injury (IRI), but the mechanism is not well understood. Tregs express CD73, the final enzyme involved in the production of extracellular adenosine, and activation of the adenosine 2A receptor (A(2A)R) on immune cells suppresses inflammation and preserves kidney function after IRI. We hypothesized that Treg-generated adenosine is required to block innate immune responses in kidney IRI and that the Treg-generated adenosine would signal through A(2A)Rs on inflammatory cells and, in an autocrine manner, on Tregs themselves. We found that adoptively transferred wild-type Tregs protected wild-type mice from kidney IRI, but the absence of adenosine generation (CD73-deficient Tregs) or adenosine responsiveness (A(2A)R-deficient Tregs) led to inhibition of Treg function. Pharmacologic stimulation of A(2A)R before adoptive transfer augmented the ability of wild-type and CD73-deficient Tregs to suppress kidney IRI. Microarray analysis and flow cytometry revealed that A(2A)R activation enhanced surface PD-1 expression on Tregs in the absence of any other activation signal. Treatment of Tregs with a PD-1 blocking antibody before adoptive transfer reversed their protective effects, even if pretreated with an A(2A)R agonist. Taken together, these results demonstrate that the simultaneous ability to generate and respond to adenosine is required for Tregs to suppress innate immune responses in IRI through a PD-1-dependent mechanism.

Rapid Apoptosis Induced by Shiga Toxin in HeLa Cells

ABSTRACT Apoptosis was induced rapidly in HeLa cells after exposure to bacterial Shiga toxin (Stx1 and Stx2; 10 ng/ml). Approximately 60% of HeLa cells became apoptotic within 4 h as detected by DNA fragmentation, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and electron microscopy. Stx1-induced apoptosis required enzymatic activity of the Stx1A subunit, and apoptosis was not induced by the Stx2B subunit alone or by the anti-globotriaosylceramide antibody. This activity was also inhibited by brefeldin A, indicating the need for toxin processing through the Golgi apparatus. The intracellular pathway leading to apoptosis was further defined. Exposure of HeLa cells to Stx1 activated caspases 3, 6, 8, and 9, as measured both by an enzymatic assay with synthetic substrates and by detection of proteolytically activated forms of these caspases by Western immunoblotting. Preincubation of HeLa cells with substrate inhibitors of caspases 3, 6, and 8 protected the cells against Stx1-dependent apoptosis. These results led to a more detailed examination of the mitochondrial pathway of apoptosis. Apoptosis induced by Stx1 was accompanied by damage to mitochondrial membranes, measured as a reduced mitochondrial membrane potential, and increased release of cytochrome c from mitochondria at 3 to 4 h. Bid, an endogenous protein known to permeabilize mitochondrial membranes, was activated in a Stx1-dependent manner. Caspase-8 is known to activate Bid, and a specific inhibitor of caspase-8 prevented the mitochondrial damage. Although these data suggested that caspase-8-mediated cleavage of Bid with release of cytochrome c from mitochondria and activation of caspase-9 were responsible for the apoptosis, preincubation of HeLa cells with a specific inhibitor of caspase-9 did not protect against apoptosis. These results were explained by the discovery of a simultaneous Stx1-dependent increase in endogenous XIAP, a direct inhibitor of caspase-9. We conclude that the primary pathway of Stx1-induced apoptosis and DNA fragmentation in HeLa cells is unique and includes caspases 8, 6, and 3 but is independent of events in the mitochondrial pathway.