M. J. Bowles

Activation antigen expression on peripheral blood neutrophils following rat small bowel transplantation: NKR-P1 is a novel antigen preferentially expressed during allograft rejection

This study used flow cytometric analyses to monitor activation antigen expression (MHC class II; interleukin-2 receptor, p55IL-2R and 3.2.3/NKR-P1 antigen) on peripheral blood neutrophils following rat small bowel transplantation. The rat 3.2.3 antigen is a member of the NKR-P1 family of natural killer (NK) cell-associated molecules, which are expressed at high levels on NK cells and lymphokine-activated killer cells, and low levels on at least one T cell subset. Peripheral blood neutrophils in normal animals express very low or undetectable levels of NKR-P1. Detectable levels of NKR-P1 were induced as early as day 1 following small bowel transplantation in all allografted animals, whereas expression was only rarely detected in isografted animals. In addition, NKR-P1 density was significantly higher in allografted animal and was maintained as rejection developed. MHC class II and p55IL-2R expression was also induced following transplantation. The mechanisms of induction and functional relevance of NKR-P1 expression on neutrophils remain to be defined. However, the concomitant increased expression of MHC class II and p55IL-2R suggest NKR-P1 to be a neutrophil activation marker and implicate a potential role for NKR-P1+ neutrophils in small bowel allograft rejection. This hypothesis is further supported by the loss of detectable peripheral blood neutrophils only with developing rejection. Flow cytometric analysis of neutrophil activation antigen expression may be useful for monitoring human small bowel transplant recipients.

Effect of anti-LFA-1 monoclonal antibody on rat small bowel allograft survival and circulating leukocyte populations.

Anti-LFA-1 monoclonal antibodies (mAb) prolong graft survival in several animal models. This study assessed the effect of an anti-LFA-1 mAb (WT.1) on small bowel allograft rejection, circulating leukocyte subsets and in vivo target cell antigen blockade. Heterotopic small bowel transplantation was performed between PVG donor and DA recipient rats. Transplanted animals received 1 mg/kg per day WT.1 on days -1, 0 (day of transplantation) and 1. Three doses of WT.1 were also administered to a group of untransplanted animals to monitor circulating leukocyte populations and in vivo binding. WT.1 prolonged recipient survival from 7 to 14 days. Peripheral leukocyte counts increased more than twofold, primarily due to marked increases in both CD4+ and CD8+ lymphocytes. Approximately 85% of WT.1 binding sites on lymphocytes and monocytes were blocked/modulated after the course of therapy. WT.1 has marked effects on circulating leukocytes and target cell binding capacities and can affect the survival of rat small bowel transplant recipients.

Effect of anti-CD4 monoclonal antibody administration on rat small bowel allograft survival and circulating leukocyte populations

Abstract This study assessed the effect of an anti‐rat CD4 monoclonal antibody (OX38) on heterotopic small bowel allograft rejection. Fully allogeneic small bowel transplants were performed in the PVG‐to‐DA‐rat strain combination. Animals received either i) short course (days ‐1, 0 and 1) of 1 mg/kg per day OX38, ii) short course of 5 mg/kg per day or iii) extended course (days ‐2, ‐1, 0, 1, 2 and twice weekly thereafter) of 1 mg/kg per day. Both the high dose (13 days) and extended low‐dose (12 days) courses prolonged graft survival compared to untreated control animals (7 days). The low‐dose, short‐course treatment had no effect. Similar regimens were given to animals that did not receive transplants and in which peripheral blood CD4+ cell counts fell to between 20 and 55% of pretreatment levels and 20–30% of binding sites were blocked. In summary, anti‐CD4 monoclonal antibody therapy delayed rejection of rat small bowel allografts; however, long‐term survival was not achieved.

An Enzyme Immunoassay for Rat Soluble MHC Class I Molecules (RT1) and the Release of Soluble Class I From Mitogenically Stimulated Mononuclear Cells

Soluble MHC class I antigens can be detected in the serum of humans and various animals and appear in the circulation shortly after liver transplantation. The precise role of these antigens is currently uncertain, but soluble MHC class I may be involved in immunomodulation. We have developed an enzyme linked immunosorbent assay for soluble rat MHC class I (RT1a) molecules and monitored the kinetics of antigen release following in vitro stimulation of splenic mononuclear cells. A 4 day DA splenocyte Con A supernatant provided a source of soluble class I antigens and was arbitrarily assigned a concentration of 1000 units/ml. Ninety six well plates were coated with a rat RT1a-specific mAb (MN4-91-6) and soluble class I binding was detected using a biotinylated mAb reactive with a monomorphic region of the rat MHC class I molecule (OX18) followed by a streptavidin-alkaline phosphatase conjugate and substrate. The intra- and interassay variations were typically less than 5% and 10% respectively, to give a working range for the assay of between 62.5 and 1000 units/ml. Mitogenic stimulation led to a progressive increase in soluble class I levels in culture supernatants. This assay will be valuable in differentiating recipient and graft responses following experimental organ transplantation.

Induction of antigraft and antirecipient antibody responses after fully allogeneic and semiallogeneic rat small bowel transplantation.

Background. Given the potential influence of alloantibodies on organ graft outcome, this study investigated the induction of antigraft and antirecipient antibodies after allogeneic and semiallogeneic rat small bowel transplantation. Methods. Fully allogeneic, unidirectional rejection and unidirectional graft-versus-host disease (GvHD) heterotopic small bowel transplantation was performed using DA, PVG, and (PVGxDA)F1 donor-recipient combinations. Serum was obtained before and at time points after transplantation and incubated with blood from untransplanted DA and PVG rats. Antibody binding to T cells was detected by whole blood flow cytometry using FITC-conjugated anti-rat IgM murine monoclonal antibody. Antibody levels were determined by reference to a standard curve of fluorescent intensity generated using a serum sample with known anti-target cell IgM activity. Data are presented as arbitrary units/ml (AU/ml). Results. In the PVG→DA combination, five of six DA recipients had detectable anti-graft (PVG) antibodies by day 4 after transplantation (mean 72 AU/ml) and all animals were positive by day 6 (976 AU/ml). Antirecipient (DA) antibodies were also induced, however, they were only apparent after 6 days in five of eight animals (90 AU/ml). Antigraft (DA) antibody responses were also induced in the DA→PVG combination (day 6–218 AU/ml), however no antirecipient (PVG) response was apparent. Transplantation induced antirecipient (DA) antibodies in the unidirectional GvHD model (day 6–90 AU/ml) and an anti-graft (PVG) response in the unidirectional rejection model (day 6–60 AU/ml). However, the latter was quantitatively lower than that generated in the PVG→DA combination (day 6–976 AU/ml). Conclusions. Antigraft and antirecipient antibody responses are simultaneously induced after fully allogeneic small bowel transplantation, despite rejection being the predominant clinical feature. Further studies are required to elucidate their influence on graft outcome.

Antimurine immunoglobulin antibody responses after the administration of murine monoclonal antibodies to rats are altered by small bowel allograft rejection.

BACKGROUNDnThis study monitored the induction of antimurine immunoglobulin antibody responses after the administration of anti-CD4 (OX38) and anti-LFA-1 (WT.1) monoclonal antibodies to DA rats.nnnMETHODSnMonoclonal antibody was administered i.v. on 3 consecutive days to untransplanted DA rats, and DA recipients of PVG small bowel allografts. Control animals received no monoclonal antibody. Antimurine immunoglobulin antibody levels in serum samples were determined by enzyme immunoassay.nnnRESULTSnNo antimurine immunoglobulin antibody was detected in untransplanted animals receiving OX38 alone. Reactivity was apparent in WT.1-treated animals, but this response was totally abrogated by the co-administration of OX38. A combination of OX38 and WT.1 had no effect on allograft recipient survival and antimurine immunoglobulin antibody responses were detected in all allograft recipients, irrespective of the treatment regimen.nnnCONCLUSIONSnAlthough OX38 inhibited the antibody response both to itself and to WT.1 in untransplanted animals, the immune reaction induced by small bowel allograft rejection overcame this inhibitory capacity.