Early chemokine cascades in murine cardiac grafts regulate T cell recruitment and progression of acute allograft rejection.

The identification of early inflammatory events after transplant in solid tissue organ grafts that may direct T cell recruitment and promote acute allograft rejection remain largely unknown. To better understand temporal aspects of early inflammatory events in vascularized organ grafts, we tested the intragraft expression of four different chemokines in heterotopically transplanted A/J (H-2(a)) and syngeneic heart grafts in C57BL/6 (H-2(b)) recipient mice from 1.5 to 48 h after transplant. Similar temporal expression patterns and equivalent levels of chemokine expression were observed in both syngeneic and allogeneic cardiac allografts during this time period. Expression of the neutrophil chemoattractant growth-related oncogene alpha (KC) was observed first and reached peak levels by 6 h after transplant and was followed by the monocyte/macrophage chemoattractant protein-1 (JE) and then macrophage inflammatory proteins 1beta and 1alpha. Administration of rabbit KC antiserum to allograft recipients within 30 min of cardiac transplantation attenuated downstream events including intra-allograft expression of the T cell chemoattractants IFN-gamma-inducible protein-10 and monokine induced by IFN-gamma, cellular infiltration into the allograft, and graft rejection. Similarly, depletion of recipient neutrophils at the time of transplantation significantly extended allograft survival from day 8 to 10 in control-treated recipients up to day 21 after transplant. These results indicate the induction of highly organized cascades of neutrophil and macrophage chemoattractants in cardiac grafts and support the proposal that early inflammatory events are required for optimal recruitment of T cells into allografts during the progression of acute rejection of cardiac allografts.

Negative regulation of CD8+ T cell function by the IFN-induced and double-stranded RNA-activated kinase PKR.

The IFN-induced and dsRNA-activated kinase (PKR) mediates the antiviral and antiproliferative effects of IFN-alpha and IFN-gamma. Despite these findings, PKR:(-/-) mice have no overt immunological phenotype. Here we tested the role of PKR in cellular immunity by determining the induction and elicitation of contact hypersensitivity in PKR:(-/-) mice, a model of T cell-mediated immunity. When compared with wild type, the magnitude of contact hypersensitivity responses in PKR:(-/-) mice were 2-fold higher and of extended duration. This was also observed when naive recipients of immune CD8(+) T cells from sensitized PKR:(-/-) and CD4(+) T cells from sensitized wild-type PKR:(+/+) or PKR:(-/-) mice were challenged with hapten, indicating a regulatory defect intrinsic to the CD8(+) T cell population. Isolated lymph node T cells from PKR:(-/-) mice were hyperproliferative during Con A-mediated stimulation. These results implicate PKR for the first time in the growth control of mature T lymphocytes and give insight into the negative regulation of CD8(+) T cell-mediated immune responses.

Inhibition of functional T cell priming and contact hypersensitivity responses by treatment with anti-secondary lymphoid chemokine antibody during hapten sensitization.

Recent studies have suggested a pivotal role for secondary lymphoid chemokine (SLC) in directing dendritic cell trafficking from peripheral to lymphoid tissues. As an extension of these studies, we examined the consequences of anti-SLC Ab treatment during Ag priming on T cell function in an inflammatory response. We used a model of T cell-mediated inflammation, contact hypersensitivity (CHS), where priming of the effector T cells is dependent upon epidermal dendritic cell, Langerhans cells, and migration from the hapten sensitization site in the skin to draining lymph nodes. A single injection of anti-SLC Ab given at the time of sensitization with FITC inhibited Langerhans cell migration into draining lymph nodes for at least 3 days. The CHS response to hapten challenge was inhibited by anti-SLC Ab treatment in a dose-dependent manner. Despite the inhibition of CHS, T cells producing IFN-gamma following in vitro stimulation with anti-CD3 mAb or with hapten-labeled cells were present in the skin-draining lymph nodes of mice treated with anti-SLC Ab during hapten sensitization. These T cells were unable, however, to passively transfer CHS to naive recipients. Animals treated with anti-SLC Ab during hapten sensitization were not tolerant to subsequent sensitization and challenge with the hapten. In addition, anti-SLC Ab did not inhibit CHS responses when given at the time of hapten challenge. These results indicate an important role for SLC during sensitization for CHS and suggest a strategy to circumvent functional T cell priming for inflammatory responses through administration of an Ab inhibiting dendritic cell trafficking.

Intraallograft chemokine RNA and protein during rejection of MHC-matched/multiple minor histocompatibility-disparate skin grafts.

Chemokines direct leukocyte recruitment into sites of tissue inflammation and may facilitate recruitment of leukocytes into allografts following transplantation. Although the expression of chemokines during rejection of MHC-disparate allografts has been examined, chemokine expression in MHC-matched/multiple minor histocompatibility Ag-disparate allografts has not been tested. The intraallograft RNA expression of several C-X-C and C-C chemokines was tested during rejection of full thickness skin grafts from B10. D2 donors on control Ig-, anti-CD4 mAb-, and anti-CD8 mAb-treated BALB/c recipients. In all recipients, two patterns of intragraft chemokine expression were observed during rejection of these grafts: 1) macrophage-inflammatory protein-1alpha, macrophage-inflammatory protein-1beta, GRO-alpha (KC), JE, and IFN-gamma-inducible protein (IP-10) were expressed at equivalent levels in allo- and isografts for 2-4 days posttransplant and then returned to low or undetectable levels; and 2) IP-10 and monokine induced by IFN-gamma (Mig) were expressed in the allografts 3 days before rejection was completed, suggesting a possible role in recruiting primed T cells into the allograft. Three days before completion of rejection, intraallograft IP-10 protein was restricted to the epidermis, whereas Mig was located in the lower dermis and associated with the intense infiltration of mononuclear cells. Treatment of B10.D2 recipients with rabbit antiserum to Mig, but not to IP-10, delayed rejection of the allografts 3-4 days. The results suggest that Mig mediates optimal recruitment of T cells into MHC-matched/multiple minor histocompatibility Ag-disparate allografts during rejection.

Early expression of interferon-gamma inducible protein 10 and monokine induced by interferon-gamma in cardiac allografts is mediated by CD8+ T cells.

BACKGROUND: Our goal was to test the intragraft mRNA expression and production of two chemokines that are potent chemoattractants for antigen-primed T cells, interferon-gamma inducible protein 10 (IP-10) and monokine-induced by IFN-gamma, (Mig), in allogeneic heart grafts. METHODS: Syngeneic or allogeneic A/J (H-2a) hearts were heterotopically transplanted to wild-type, CD4-/-, CD8alpha-/-, or IFN-gamma-/- C57BL/6 (H-2b) recipients. To test expression of IP-10 and Mig, grafts were removed 1-8 days posttransplant for RNA isolation and Northern blot analysis. To test the potential recipient leukocyte populations mediating intraallograft expression of IP-10 and Mig, recipients were treated with anti-NK 1.1, anti-CD4, and/or anti-CD8 monoclonal antibodies before transplantation. RESULTS: Allogeneic heart grafts transplanted to wild-type, but not IFN-gamma-/-, recipients expressed IP-10 and Mig at day +2 posttransplant that increased thereafter until rejection was completed. Expression of IP-10 and Mig in isografts was low or undetectable. Cardiac allografts from CD8+ T cell depleted, but not NK cell or CD4+ T cell depleted, recipients had low to undetectable expression of IP-10 and Mig on day +2 posttransplant. Similarly, cardiac allografts from CD8-/-, but not CD4-/-, recipients had low to undetectable expression of IP-10 and Mig on day +2 posttransplant. CONCLUSIONS: Early intraallograft expression of Mig and IP-10 during primary rejection of cardiac allografts is dependent on the activities of recipient CD8+ T cells.

T cell infiltration into class II MHC-disparate allografts and acute rejection is dependent on the IFN-gamma-induced chemokine Mig.

Direct evidence that cytokines with chemoattractant properties for leukocytes, chemokines, recruit alloantigen-primed T cells into transplanted allografts has been lacking. We present evidence that neutralization of a single chemokine inhibits T cell infiltration into class II MHC-disparate murine allografts and acute rejection. The chemokines IFN-gamma-inducible protein-10 and monokine induced by IFN-gamma (Mig) are expressed in allogeneic skin grafts during the late stages of acute rejection. Survival of class II MHC-disparate B6.H-2bm12 allografts is prolonged from day 14 to day 55 posttransplant when C57BL/6 recipients are given a short course treatment with an antiserum to Mig. This treatment also inhibits T cell and macrophage infiltration into the allografts. B6.H-2bm12 allografts are also not rejected by IFN-gamma-/- C57BL/6 recipients. Injection of Mig directly into B6.H-2bm12 grafts on IFN-gamma-deficient recipients restores T cell infiltration and rejection. Therefore, the inability of IFN-gamma-deficient recipients to reject the class II MHC-disparate allografts is due to the lack of intraallograft Mig production and alloantigen-primed T cell recruitment to the graft. These results indicate for the first time the potential utility of chemokine neutralization strategies in preventing T cell infiltration into allografts and abrogating acute rejection.