Abrogation by rapamycin of accelerated rejection in sensitized rats by inhibition of alloantibody responses and selective suppression of intragraft mononuclear and endothelial cell activation, cytokine production, and cell adhesion.

This study evaluated the efficacy and mode of action of rapamycin (RPM) in a model of accelerated (24-hr) rejection of LBNF1 cardiac allografts in specifically sensitized LEW rats. RPM treatment (0.25 mg/kg/day i.p.) between the day of sensitizing skin grafts (day -7) and subsequent heart (day 0) transplantation (Tx), abrogated fulminant rejection and prolonged cardiac allograft survival to 46 +/- 22 days (mean +/- SD, P < 0.0001). The delayed introduction of RPM until day -2 or day -1 was equally effective, whereas treatment initiated after cardiac Tx was ineffectual. Untreated accelerated rejection was associated with strong production of circulating IgM, whereas an IgG alloantibody response was not detected until after rejection was complete. RPM therapy (day -7 to -1) diminished this systemic IgM response and prevented the switch from IgM to IgG alloantibody production. Immunohistologic evaluation at 24 hr after cardiac Tx showed that compared with untreated hosts RPM treatment largely abolished intragraft cellularity, and was associated with decreased mononuclear and endothelial cell activation. Specifically, Ia and ICAM-1 upregulation was abolished, and no cells elaborating IL-2 or IFN-gamma were detected. In addition, RPM treatment prevented intragraft production of the proinflammatory cytokines IL-1 beta, IL-6, and IL-8. The effects of RPM therapy on recipient cellular responses were evaluated in vitro by mixed lymphocyte reaction. Surprisingly, the donor-specific proliferative response of cells from RPM-treated hosts at 1 or 7 days after Tx was markedly increased, compared with cells from rejecting, untreated controls, and bioassay of IL-2 within supernatants of MLR cultures showed comparable levels of IL-2 in both groups. The effects of RPM upon adhesion properties of lymph node lymphocytes were also tested in an in vitro binding assay. The binding of naive cells to sections of cardiac allografts collected from RPM-treated hosts at 24 hr post-Tx was decreased compared with that in untreated recipients. Interestingly, the binding of mononuclear cells to high endothelial venules of peripheral lymph nodes in RPM-treated hosts remained relatively high. Thus, treatment with RPM prevents and/or erases the sensitization, which otherwise leads to accelerated allograft rejection. Abrogation of allograft injury by RPM was associated with profound and long-lasting depression of host IgM and IgG alloantibody responses in the circulation, and selective downregulation of host cellular immunity and endothelial activation at the graft site. In contrast, antigen alloreactivity and endothelial adhesivity in peripheral lymphoid tissues were spared, indicating novel and potent selective effects of RPM therapy in allograft recipients.

Schmidbauer G ，Hancock WW ，Wasowska B ，Badger AM ，Kupiec-Weglinski JW ... -  《TRANSPLANTATION》

Cytokine and alloantibody networks in long term cardiac allografts in rat recipients treated with rapamycin.

Treatment with rapamycin (RPM) prevents accelerated rejection of (LEW x BN)F1 cardiac allografts in LEW rats presensitized with BN skin grafts. This study analyzed the influence of RPM on cytokine (IL-2, IL-4, IL-10, and IL-12) and alloantibody networks in this model. Accelerated (24-h) rejection was associated with strong expression of intragraft IL-2 and IL-12 (p40) mRNAs, which reached maximal levels 3 to 6 h post-transplantation. IL-4 and IL-10 mRNAs were readily detectable throughout the observation period. RPM therapy abrogated rejection at 24 h and prolonged cardiac allograft survival to about 50 days. This effect was correlated with a profound initial depression of IL-2 mRNA; delayed expression of IL-2 mRNA was detected in well functioning grafts at > 20 days. In RPM-treated hosts, expression of IL-12 (p40) mRNA was low at the early time points (6-24 h), but prominent in long term grafts. The expression of both IL-4 and IL-10 mRNAs was preserved in RPM-conditioned hosts. Immunohistologic analysis of long term allografts revealed an interstitial cellular infiltrate and areas of intimal proliferation within small arteries indicative of early transplant arteriosclerosis. Analysis of cytokine proteins showed dense labeling of mononuclear and some endothelial cells for IL-4 and IL-12 (p70), but not for IL-2 or IFN-gamma alloantibody in the early post-transplant period. However, an increase in circulating and intragraft IgM and, to a lesser extent, IgG, primarily of the IgG2b subclass, was evident in long term recipients. Thus, RPM treatment reduces, but does not completely inhibit, the expression of Th1-type and preserves the expression of Th2-type cytokines. The demonstration of IL-12 in long term allografts after RPM therapy may reflect late activation of macrophages that, coupled with the appearance of IgG2b, may contribute to the chronic rejection of cardiac allografts.

Wasowska B ，Wieder KJ ，Hancock WW ，Zheng XX ，Berse B ，Binder J ，Strom TB ，Kupiec-Weglinski JW ... -  《JOURNAL OF IMMUNOLOGY》

CD4 mAb therapy modulates alloantibody production and intracardiac graft deposition in association with selective inhibition of Th1 lymphokines.

The accelerated (24 h) rejection of (LEWxBN)F1 cardiac allografts (Tx) in LEW rats sensitized with BN skin grafts, is abrogated with CD4 mAb (BWH-4) administration between skin (day -7) and heart (day 0) transplantation (Tx survival ca. 11 days, p < 0.0001). This study analyzed the effects of CD4-targeted therapy upon host IgG and IgM alloantibody (allo-Ab) within the serum by two-color flow cytometry, and within the Tx, by immunohistology. These data were correlated with mRNA and protein production profiles of Th1 (IL-2, IFN-gamma) vs Th2 (IL-4) specific cytokines (polymerase chain reaction and/or immunohistology). Skin grafts elicited a strong systemic IgM allo-Ab response, which peaked at the time of cardiac Tx rejection at 24 h. It was associated with extensive deposits of IgM on Tx endothelium. Treatment with BWH-4 mAb diminished circulating IgM allo-Ab levels, and only low levels of IgM could be detected at the Tx site. Conversely, the low circulating IgG allo-Ab levels during rejection at 24 h in untreated recipients were accompanied by a strong labeling for intra-Tx IgG. BWH-4 mAb therapy did not prevent totally the switch of the IgM to IgG, but the IgG allo-Ab response was earlier, less intense and more transient than in untreated recipients. Accelerated rejection triggered sequential lymphokine mRNA expression in cardiac Tx, with the peak of transcription for IL-2 (6-12 h) preceding that for IL-4 (24 h). Interestingly, although CD4 targeted therapy virtually ablated the induction of IL-2 mRNA, it preserved transcription of the IL-4 gene. BWH-4 mAb therapy decreased otherwise abundant intra-Tx IL-2 and IFN-gamma, but allowed a vigorous elaboration of IL-4, confirming the translation of mRNA to the protein in vivo. Thus, CD4 mAb-mediated abrogation of accelerated cardiac Tx injury correlates with suppression of Th1 responses (depressed IL-2 and IFN-gamma production), but sparing of the Th2 function (enhanced IL-4 elaboration). Indeed, CD4 mAb-induced allo-Ab depression and immunosuppressive effects may reflect selective targeting of proinflammatory Th1-like cells and the multifaceted effects of IL-4 produced by unopposed Th2-like cells.

Kupiec-Weglinski JW ，Wasowska B ，Papp I ，Schmidbauer G ，Sayegh MH ，Baldwin WM 3rd ，Wieder KJ ，Hancock WW ... -  《JOURNAL OF IMMUNOLOGY》

Rapamycin treatment depresses intragraft expression of KC/MIP-2, granzyme B, and IFN-gamma in rat recipients of cardiac allografts.

Rapamycin (RPM) treatment prevents accelerated rejection of cardiac allografts in sensitized rats. The prominent feature of this brisk 24-h rejection, which includes a panoply of both cellular and humoral host immune responses, is a massive infiltration of rejecting grafts with neutrophils. In this study we tested the hypothesis that RPM-mediated therapeutic effects on accelerated rejection may be linked to decreased expression of protein encoded by gro/melanoma-growth stimulatory activity gene (KC) and macrophage inflammatory protein-2 (MIP-2) genes, the operational rat homologues of the human intercrine-alpha cytokines with proinflammatory IL-8-like neutrophil activation/chemotactic properties. The induction of these genes was then correlated with mRNA profiles encoding for Th1-selective IFN-gamma and CTL-specific granzyme B proteins. Northern blot analysis of RNA from cardiac allografts of sensitized untreated recipients, revealed maximal levels of KC and MIP-2 mRNA at 3 to 6 h after transplantation. In contrast, IFN-gamma mRNA, which was at most very weakly expressed at 3 h, peaked between 6 to 12 h. As with IFN-gamma, granzyme B transcripts were undetectable at 3 h, but peaked around the time of actual graft rejection at 24 h. RPM therapy abrogated accelerated rejection and prolonged cardiac allograft survival to ca. 46 days. This effect was associated with markedly reduced expression of KC and MIP-2 mRNA in the first 24 h as well as at 7 and 34 days after transplantation. Moreover, RPM completely blocked intragraft appearance of granzyme B and IFN-gamma mRNA in long term cardiac allografts. Immunohistologic analysis has revealed that accelerated rejection was associated with extensive neutrophil infiltration, which peaked at 18 to 24 h. At this time, leukocytes and endothelium were intensely stained for IL-8 and IFN-gamma antibodies. In contrast, the allografts from RPM-treated hosts showed essentially no neutrophil infiltration and minor, focal staining for IL-8 and IFN-gamma. This study demonstrates an association between the early expression of genes for proinflammatory IL-8-dependent neutrophil chemotactic activity, and later expression of genes associated with activation/effector activity of CTL and NK cells. It also documents a novel effect of RPM in vivo, which results in the suppression of intragraft IL-8-like and CTL-dependent mRNA/protein production and diminished neutrophil infiltration; these may contribute to the striking efficacy of RPM therapy in sensitized graft recipients.

Wieder KJ ，Hancock WW ，Schmidbauer G ，Corpier CL ，Wieder I ，Kobzik L ，Strom TB ，Kupiec-Weglinski JW ... -  《JOURNAL OF IMMUNOLOGY》

Oral, but not intravenous, alloantigen prevents accelerated allograft rejection by selective intragraft Th2 cell activation.

We studied the mechanisms by which oral or intravenous administration of allogeneic splenocytes prevents sensitization by skin allografts and development of accelerated rejection of subsequent cardiac allografts. LEW rats were sensitized with BN skin allografts 7 days prior to receiving heterotopic (LEW x BN)F1 vascularized cardiac allografts. While unsensitized cardiac allografts are rejected on days 6-8, control sensitized grafts were rejected within 24 to 48 hr. Oral administration of BN splenocytes during the sensitization phase (between skin and heart grafting) has been found to prevent accelerated allograft rejection and prolong cardiac allograft survival to 7 days. An alternative route of antigen exposure, specifically intravenous administration of BN splenocytes (50 x 10(6) daily for 5 days starting on the day of skin grafting), also prevented accelerated cardiac allograft rejection and prolonged allograft survival to 9 +/- 1 days (n = 5). Immunoperoxidase studies of cardiac allografts harvested 24-48 hr posttransplant showed that, when compared with sensitized controls, animals that received oral splenocytes had reduced deposition of IgG (end-point titer of 1/1000 vs. 1/4000), IgM (1/1000 vs. 1/16000), C3 (1/4000 vs. 1/16000), and fibrin (1/4000 vs. 1/16000). There was also decreased infiltration by macrophages (18 +/- 8 vs. 37 +/- 8 cells/HPF, P < 0.01), T cells (5 +/- 3 vs. 19 +/- 7, P < 0.01), and IL-2R+ T cells (5 +/- 3 vs. 15 +/- 4, P < 0.01), and a significant reduction in the numbers and extent of intragraft mononuclear cells stained with antibodies to IL-1, IL-2, IL-6, IL-8, IFN-gamma, and TNF-alpha. In contrast, these grafts showed markedly increased IL-4 staining (including most mononuclear and all endothelial cells), as compared with control grafts (< 20% of mononuclear cells and only focal endothelial staining). Immunoperoxidase studies of cardiac allografts harvested from rats receiving intravenous splenocytes also showed markedly reduced humoral deposits and cellular infiltrates, comparable to that found in the oral splenocytes-treated group, but showed significantly different cytokine expression. In particular, some intragraft mononuclear cell labeling for IFN-gamma remained, and IL-4 staining was not increased relative to control grafts. Attempts were then made to abrogate spleen cell-induced prolongation of cardiac allograft survival by daily injections of CD4 monoclonal antibody (BWH-4 mAb, 700 micrograms) from the time of cardiac transplantation, therapy previously shown unable to prolong cardiac survival in this model when commenced after skin graft-induced sensitization has occurred.(ABSTRACT TRUNCATED AT 400 WORDS)

Hancock WW ，Sayegh MH ，Kwok CA ，Weiner HL ，Carpenter CB ... -  《TRANSPLANTATION》