Chemokines provide signals for activation and recruitment of effector cells into sites of inflammation, acting via specific G protein–coupled receptors. However, in vitro data demonstrating the presence of multiple ligands for a given chemokine receptor, and often multiple receptors for a given chemokine, have led to concerns of biologic redundancy. Here we show that acute cardiac allograft rejection is accompanied by progressive intragraft production of the chemokines interferon (IFN)-γ–inducible protein of 10 kD (IP-10), monokine induced by IFN-γ (Mig), and IFN-inducible T cell α chemoattractant (I-TAC), and by infiltration of activated T cells bearing the corresponding chemokine receptor, CXCR3. We used three in vivo models to demonstrate a role for CXCR3 in the development of transplant rejection. First, CXCR3-deficient (CXCR3−/−) mice showed profound resistance to development of acute allograft rejection. Second, CXCR3−/− allograft recipients treated with a brief, subtherapeutic course of cyclosporin A maintained their allografts permanently and without evidence of chronic rejection. Third, CXCR+/+ mice treated with an anti-CXCR3 monoclonal antibody showed prolongation of allograft survival, even if begun after the onset of rejection. Taken in conjunction with our findings of CXCR3 expression in rejecting human cardiac allografts, we conclude that CXCR3 plays a key role in T cell activation, recruitment, and allograft destruction.

An allograft is often considered an immunologically inert playing field on which host leukocytes assemble and wreak havoc. However, we demonstrate that graft-specific physiologic responses to early injury initiate and promulgate destruction of vascularized grafts. Serial analysis of allografts showed that intragraft expression of the three chemokine ligands for the CXC chemo-kine receptor CXCR3 was induced in the order of interferon (IFN)-γ–inducible protein of 10 kD (IP-10, or CXCL10), IFN-inducible T cell α-chemoattractant (I-TAC; CXCL11), and then monokine induced by IFN-γ (Mig, CXCL9). Initial IP-10 production was localized to endothelial cells, and only IP-10 was induced by isografting. Anti–IP-10 monoclonal antibodies prolonged allograft survival, but surprisingly, IP-10–deficient (IP-10−/−) mice acutely rejected allografts. However, though allografts from IP-10+/+ mice were rejected by day 7, hearts from IP-10−/− mice survived long term. Compared with IP-10+/+ donors, use of IP-10−/− donors reduced intragraft expression of cytokines, chemokines and their receptors, and associated leukocyte infiltration and graft injury. Hence, tissue-specific generation of a single chemokine in response to initial ischemia/reperfusion can initiate progressive graft infiltration and amplification of multiple effector pathways, and targeting of this proximal chemokine can prevent acute rejection. These data emphasize the pivotal role of donor-derived IP-10 in initiating alloresponses, with implications for tissue engineering to decrease immunogenicity, and demonstrate that chemokine redundancy may not be operative in vivo.

The transient expression of many different genes is mediated by the inducible transcription factor p50‐p65 NF kappa B, which in turn is regulated by complex formation with its inhibitor I kappa B alpha. We describe here that in porcine aortic endothelial cells, either IL‐1 alpha, TNF alpha or LPS upregulates an inhibitor of NF kappa B which we refer to as ECI‐6. ECI‐6 is by structural and functional criteria an I kappa B alpha protein, the porcine homologue of MAD‐3, pp40 and RL/IF‐1. We have studied the promoter of the ECI‐6/I kappa B alpha gene and provide three lines of evidence that its expression is directly regulated by NF kappa B. First, the 5′ regulatory region of ECI‐6/I kappa B alpha contains two sites that bind NF kappa B in electrophoretic mobility shift assays. Second, expression following transfection of an ECI‐6/I kappa B alpha promoter‐luciferase reporter construct is dependent on a co‐transfected NF kappa B‐p65 subunit. Third, pretreatment of endothelial cells with antioxidants, agents that inhibit activation of NF kappa B, inhibit the expression of ECI‐6/I kappa B alpha. We conclude that the regulated expression of ECI‐6/I kappa B alpha could represent a novel feedback mechanism by which NF kappa B downregulates its own activity after transient activation of target genes has been achieved.

The activity of the transcription factor NF-B is thought to be regulated mainly through cytoplasmic retention by IB molecules. Here we present evidence of a second mechanism of regulation acting on NF-B after release from IB. In endothelial cells this mechanism involves phosphorylation of the RelA subunit of NF-B through a pathway involving activation of protein kinase C (PKC) and p21 ras . We show that transcriptional activity of RelA is dependent on phosphorylation of the N-terminal Rel homology domain but not the C-terminal transactivation domain. Inhibition of phosphorylation by dominant negative mutants of PKC or p21 ras results in loss of RelA transcriptional activity without interfering with DNA binding. Raf/MEK, small GTPases, phosphatidylinositol 3-kinase, and stress-activated protein kinase pathways are not involved in this mechanism of regulation.The NF-B/Rel family of dimeric transcription factors is involved in the immediate early transcription, i.e. independent of protein synthesis, of a large array of genes induced by mitogenic or pathogen-associated stimuli. In its active form, NF-B is a nuclear homo-or heterodimeric complex of a number of different Rel family members. The canonical and most abundant form of NF-B is composed of a 50-kDa (p50, or NFB1) and a 65-kDa (p65, or RelA) subunit. Both subunits can form homodimers as well as heterodimers with other members of the Rel family i.e. c-Rel (Rel), p52 (NFB2), and RelB (1). All members of the Rel family exhibit extensive sequence similarity in their N-terminal region referred to as the Rel homology domain (RHD) 1 responsible for DNA binding and formation of Rel dimers. Only RelA, Rel, and RelB carry a transcription activating domain, and thus only dimers containing one of these proteins activate the transcription of NF-B-dependent genes efficiently. With respect to transcription activation, the RelA subunit appears to have the highest activity.In most unstimulated cells, NF-B is constitutively retained in the cytoplasm by inhibitory proteins of the IB family, namely IB␣, IB␤, IB␥, p100, p105, and IB⑀ (2). Formation of NF-B⅐IB complexes masks the nuclear localization signal sequence present in NF-B molecules and thus prevents their nuclear translocation. One of the key events in the activation of NF-B is the liberation of functional NF-B dimers from IB, which results in the translocation of NF-B to the nucleus. Cytoplasmic release of NF-B dimers involves site-specific phosphorylation of IB by kinases of the IB signalosome (3-6), ubiquitination (7), and subsequent proteolytic degradation by the 26 S proteasome pathway (8). Upon nuclear import and binding to specific decameric recognition motifs, which are reflected by the consensus GGGRNNYYCC (where R represents A or G and Y represents C or T), NF-B dimers function as transcriptional activators. IB␣ (9), IB␤, and p105 (10) have been implicated in the inhibition of DNA binding of NF-B complexes. However, there have been several reports showing that NF-B transcriptional activity can be blocked withou...

IntroductionMononuclear cell recruitment to an allograft is a classic hallmark of cellular rejection. At least in broad terms, such leukocyte recruitment from the vascular pool across activated endothelial cells and into tissues is now reasonably well understood (1). Thus, leukocytes roll along selectin-expressing endothelium adjacent to a chemoattractant source, attach more firmly, change shape, migrate between adjacent endothelial cells as a result of integrin and other adhesion molecule binding, and migrate through extravascular tissues along chemotactic gradients to reach their destination. The latter chemokine/chemokine receptor phase is the least understood, with little in vivo data available. However, given the burgeoning field of chemokine biology, dissecting which molecules are generated in a given inflammatory setting, and especially the nature of chemokine receptors responsible for leukocyte recruitment, might well prove key to developing better therapeutic strategies for the prevention and treatment of allograft rejection. The current literature on chemokine receptor expression in organ transplants is limited to 2 papers noting expression of CXCR4 (ref. The current studies involve serial analysis of intragraft chemokine and chemokine receptor expression within completely MHC-mismatched mouse cardiac allografts. On the basis of our initial data, in which several chemokine receptors and their ligands were associated with host mononuclear cell infiltration, we undertook a detailed analysis of the significance of 1 of the more highly expressed chemokine receptors, CCR1 (4), which binds RANTES, macrophage inflammatory protein 1-alpha (MIP-1α), and various monocyte chemoattractant proteins (MCPs). Our studies demonstrate that compared with control CCR1 +/+ mice, CCR1 -/-mice show significantly delayed, or in some cases an absence of, acute or chronic rejection, such that targeting of CCR1 may eventually prove of therapeutic significance clinically. 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-...

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.

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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-1α), CCR2 (MCP-1), CCR5 (RANTES, MIP-1α, MIP-1β), 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-γ and TNF-α. Lastly, using recombinant proteins and transfected cells that overexpressed CCR1, we demonstrated that MIP-1α, 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.