Vilmos Csizmadia

Targeting of the chemokine receptor CCR1 suppresses development of acute and chronic cardiac allograft rejection

Although mononuclear cell infiltration is a hallmark of cellular rejection of a vascularized allograft, efforts to inhibit rejection by blocking leukocyte-endothelial cell adhesion have proved largely unsuccessful, perhaps in part because of persistent generation of chemokines within rejecting grafts. We now provide, to our knowledge, the first evidence that in vivo blockade of specific chemokine receptors is of therapeutic significance in organ transplantation. Inbred mice with a targeted deletion of the chemokine receptor CCR1 showed significant prolongation of allograft survival in 4 models. First, cardiac allografts across a class II mismatch were rejected by CCR1(+/+) recipients but were accepted permanently by CCR1(-/-) recipients. Second, CCR1(-/-) mice rejected completely class I- and class II-mismatched BALB/c cardiac allografts more slowly than control mice. Third, levels of cyclosporin A that had marginal effects in CCR1(+/+) mice resulted in permanent allograft acceptance in CCR1(-/-) recipients. These latter allografts showed no sign of chronic rejection 50-200 days after transplantation, and transfer of CD4(+) splenic T cells from these mice to naive allograft recipients significantly prolonged allograft survival, whereas cells from CCR1(+/+) mice conferred no such benefit. Finally, both CCR1(+/+) and CCR1(-/-) allograft recipients, when treated with a mAb to CD4, showed permanent engraftment, but these allografts showed florid chronic rejection in the former strain and were normal in CCR1(-/-) mice. We conclude that therapies to block CCR1/ligand interactions may prove useful in preventing acute and chronic rejection clinically.

Beneficial effects of targeting CCR5 in allograft recipients.

BACKGROUND The chemokine receptor, CCR5, and its three high-affinity ligands, macrophage inflammatory protein- (MIP) 1alpha, MIP-1beta, and regulated on activation normal T cell expressed and secreted (RANTES), are expressed by infiltrating mononuclear cells during the rejection of clinical and experimental organ allografts, although the significance of these molecules in the pathogenesis of rejection has not been established. METHODS We studied intragraft events in four allograft models. First, we studied cardiac transplants in fully MHC-mismatched mice that were deficient in CCR5 or two of its ligands, MIP-1alpha or RANTES. Second we tested the effects of a neutralizing rat anti-mCCR5 monoclonal antibody on allograft survival. Third we assessed whether a subtherapeutic course of cyclosporine would potentiate enhance survival in CCR5-deficient recipients. Finally, we tested the effect of targeting CCR5 in a class II-mismatched model. RESULTS Whereas mice deficient in expression of MIP-1alpha or RANTES reject fully MHC-mismatched cardiac allografts normally, CCR5-/- mice, or CCR5+/+ mice treated with a neutralizing mAb to mCCR5, show enhanced allograft survival. MHC class II-disparate mismatched are permanently accepted in CCR5-/- but not CCR5+/+ recipients. Finally, the beneficial effects of targeting of CCR5 are markedly synergistic with the effects of cyclosporine, resulting in permanent engraftment without development of chronic rejection. CONCLUSIONS We conclude that CCR5 plays a key role in the mechanisms of host T cell and macrophage recruitment and allograft rejection, such that targeting of CCR5 clinically may be of therapeutic significance.

Chemokines and their receptors in allograft rejection

Despite current recognition of over 40 chemokines and more than 18 chemokine receptors, understanding of their role in transplant immunobiology and transplant rejection is extremely limited and fragmentary. Recent literature has demonstrated the presence of chemokines and their receptors in transplants and some studies demonstrate important functional roles.

Targeted deletion of CX3CR1 reveals a role for fractalkine in cardiac allograft rejection

Fractalkine (Fk) is a structurally unusual member of the chemokine family. To determine its role in vivo, we generated mice with a targeted disruption of CX3CR1, the receptor for Fk. CX3CR1–/– mice were phenotypically indistinguishable from wild-type mice in a pathogen-free environment. In response to antibody-induced glomerulonephritis, CX3CR1–/– and CX3CR1+/+ mice had similar levels of proteinuria and injury. CX3CR1–/– and CX3CR1+/+ mice also developed similar levels of disease in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis. We performed heterotopic MHC class I/II cardiac transplants from BALB/c mice into C57BL/6 mice. In the absence of cyclosporin A (CsA), there was no difference in graft survival time between CX3CR1–/– and CX3CR1+/+ recipient mice. However, in the presence of subtherapeutic levels of CsA, graft survival time was significantly increased in the CX3CR1–/– mice. Characterization of cells infiltrating the grafts revealed a selective reduction in natural killer cells in the CX3CR1–/– recipients in the absence of CsA and a reduction in macrophages, natural killer cells, and other leukocytes in the presence of CsA. We conclude that Fk plays an important role in graft rejection. The development of CX3CR1 antagonists may allow reductions in the doses of immunosuppressive drugs used in transplantation.

Lack of chemokine receptor CCR1 enhances Th1 responses and glomerular injury during nephrotoxic nephritis

During the development of nephrotoxic nephritis (NTN) in the mouse, we find that a variety of chemokines and chemokine receptors are induced: CCR1 (RANTES, MIP-1alpha), CCR2 (MCP-1), CCR5 (RANTES, MIP-1alpha, MIP-1beta), CXCR2 (MIP-2), and CXCR3 (IP-10). Their timing of expression indicated that CXCR2 and CCR1 are probably important in the neutrophil-dependent heterologous phase of the disease, whereas CCR1, CCR2, CCR5, and CXCR3 accompany the subsequent mononuclear cell infiltration characteristic of autologous disease. We therefore assessed the role of CCR1 in NTN using CCR1(-/-) mice. We found that neutrophil accumulation in CCR1(-/-) mice was comparable to that in wild-type animals but that renal recruitment of CD4(+) and CD8(+) T cells and macrophages increased significantly. Moreover, CCR1(-/-) mice developed more severe glomerulonephritis than did controls, with greater proteinuria and blood urea nitrogen, as well as a higher frequency of crescent formation. In addition, CCR1(-/-) mice showed enhanced Th1 immune responses, including titers of antigen-specific IgG2a antibody, delayed-type hypersensitivity responses, and production of IFN-gamma and TNF-alpha. Lastly, using recombinant proteins and transfected cells that overexpressed CCR1, we demonstrated that MIP-1alpha, but not RANTES, bound CCR1 and induced cell chemotaxis. Thus, rather than simply promoting leukocyte recruitment during NTN, CCR1 expression profoundly alters the effector phase of glomerulonephritis. Therapeutic targeting of chemokine receptors may, on occasion, exacerbate underlying disease.

Antagonizing the α4β1 Integrin, but Not α4β7, Inhibits Leukocytic Infiltration of the Central Nervous System in Rhesus Monkey Experimental Autoimmune Encephalomyelitis

The immune system is characterized by the preferential migration of lymphocytes through specific tissues (i.e., tissue tropism). Tissue tropism is mediated, in part, by the α4 integrins expressed by T lymphocytes. The α4β1 integrin mediates migration of memory T lymphocytes into the CNS, whereas the α4β7 integrin mediates migration preferentially into gastrointestinal tissue. This paradigm was established primarily from investigations in rodents; thus, the objective of this investigation was to determine if blocking the α4β7 integrin exclusively would affect migration of T lymphocytes into the CNS of primates. The effects of the dual α4β1 and α4β7 antagonist natalizumab were compared with those of the α4β7 antagonist vedolizumab on experimental autoimmune encephalomyelitis in the rhesus monkey. Animals received an initial i.v. bolus of placebo, natalizumab (30 mg/kg), or vedolizumab (30 mg/kg) before intracutaneous immunization with recombinant human myelin oligodendrocyte glycoprotein and then Ab once weekly thereafter. Natalizumab prevented CNS inflammation and demyelination significantly (p < 0.05), compared with time-matched placebo control animals, whereas vedolizumab did not inhibit these effects, despite saturating the α4β7 integrin in each animal for the duration of the investigation. These results demonstrate that blocking α4β7 exclusively does not inhibit immune surveillance of the CNS in primates.

Differential NF-kappaB and IkappaB gene expression during development of cardiac allograft rejection versus CD154 monoclonal antibody-induced tolerance.

BACKGROUND The Rel/NF-kappaB transcription factor pathway, regulated by IkappaB proteins, is considered central to immune responses, although there are surprisingly few in vivo data concerning alloresponses. METHODS We undertook analysis of NF-kappaB and IkappaB mRNA intracardiac allograft expression, and NF-kappaB nuclear translocation, during acute rejection versus CD154 monoclonal antibody (mAb)-induced tolerance induction in fully MHC-disparate mice. RESULTS Intragraft expression of all nine NF-kappaB and IkappaB genes increased during development of rejection, and nuclear translocation of p50, p52, and p65 was detected. CD154 mAb therapy decreased mRNA levels of all nine NF-kappaB and IkappaB genes, and impaired nuclear translocation of p50, p52, and p65 NF-kappaB proteins. However, prolonged survival could not be induced by CD154 mAb in p50- or p52-deficient allograft recipients, indicating an absolute requirement for expression of these genes in CD154 mAb-induced tolerance. CONCLUSIONS We conclude that, whereas blanket approaches to NF-kappaB suppression are unlikely to be effective strategies for tolerance induction, a better understanding of the roles of individual NF-kappaB and IkappaB genes may allow development of more precise and effective therapies.

Induction of RIP-2 kinase by proinflammatory cytokines is mediated via NF-κB signaling pathways and involves a novel feed-forward regulatory mechanism

The transcription factor NF-κB (nuclear factor κB) is a central mediator of inflammatory and apoptotic signaling in the cell. The protein kinase RIP-2 is a member of the CARD protein family (caspase activation and recruitment domain, also known as CARD3, Ripk2, CARDIAK, RICK, and CCK), and has been shown to be an activator of NF-κB. In this study, it was demonstrated by transcriptional profiling and protein expression analysis that the inflammatory cytokines TNF-α, IL-1β, and IFN-γ induced RIP-2 transcription and translation in endothelial cells. Two mechanistically distinct inhibitors of NF-κB signaling, sulfasalazine (NF-κB inhibitor) and WY-14643 [PPARα (peroxisome proroliferator-activated receptor α) agonist] that interfere with the transcription factor RELA (p65), suppressed TNF-α induced RIP-2 gene expression, which indicated that NF-κB signaling was involved in the cytokine-induced transcriptional activation of RIP-2 gene expression. Consistent with these observations, multiple NF-κB response elements were found in the upstream regions of the human and mouse RIP-2 genes. NF-κB-mediated regulation of RIP-2 gene and protein expression suggests an additional step in the regulation of NF-κB function as RIP-2 has been shown to positively modulate NF-κB by binding IKKγ (IκB kinase γ), a component of the IKK complex. These findings support a positive feed-forward mechanism of NF-κB regulation that involves NF-κB-dependent induction of RIP-2 transcription and a subsequent increase in RIP-2 protein levels in response to inflammatory cytokines. Elevated RIP-2 protein levels are then available to promote NF-κB function via interaction with IKKγ. RIP-2 is the first reported NF-κB-dependent protein kinase that positively regulates NF-κB activity.

Detection of Apoptosis in Tissue Sections

TUNEL-based assays were used to demonstrate the presence of apoptotic cells in tissue sections derived from target tissues of animal models of different diseases. Emphasis was placed on tissue preparation and fixation, as these are crucial to successful histological staining. The protocol suggested here facilitates not only the reliable detection of TUNEL-positive cells but the immunodetection of different proteins in these cells and the surrounding tissues by DAB or fluorescence-based immunostaining.