T cell activation in the presence of cyclosporine in three in vivo allograft models.

The effect of cyclosporine on a systemic graft-versus-host reaction, cardiac allograft rejection, and a local host-versus-graft reaction in the rat were examined in detail. Therapeutic levels of CsA did not inhibit the early stages of lymphocyte activation but did prevent maturation of the immune response to full effector function--viz., graft rejection or clinical GVH disease. In all three models the phenotype changes in T cells associated with the early stages of activation--i.e., induction of receptors for interleukin 2 (IL-R), induction of MHC class II expression, and coexpression of CD4 and CD8 glycoproteins--were not inhibited by CsA. In the GVH and HVG reactions lymphocyte activation proceeded as far as DNA synthesis. In the systemic GVH model animals showed no sign of GVHD for as long as CsA was administered, but withdrawal of the drug resulted in accelerated lethal GVHD.

Mechanism of action of cyclosporine in preventing cardiac allograft rejection. I. Rate of entry of lymphocytes from the blood, fibrin deposition, and expression of Ia antigens on infiltrating cells.

The rate of entry of 3H-leucine-labeled lymphocytes was monitored in cyclosporine (CsA) treated or untreated rats that had received a cardiac allograft 5 days previously. In the untreated recipients there was a preferential localization of labeled cells in the graft heart compared with the native heart, which was evident within the first hour as well as at 3 and 24 hr after injection. In CsA-treated recipients, the rate and extent of entry of lymphocytes in the graft heart was not substantially different from the native heart. Fibrin deposition within allografts, thought to be a consequence of T cell activation, was substantially reduced by CsA treatment of the recipients. A double immunoenzyme staining technique was used to identify Ia-positive macrophages and Ia-positive T cells in cryostat sections of grafts: although the number of macrophages and T cells was reduced in CsA treated recipients, there was no difference in the extent to which these cells were Ia-positive.

Mechanism of action of cyclosporine in preventing cardiac allograft rejection. II. Graft tissue levels of prostacyclin and thromboxane.

Four cyclo-oxygenase products (COP) of arachidonate were measured in tissue homogenates of rat cardiac allografts at intervals after transplantation. In rejecting graft tissue, there was a progressive decrease in the levels of prostaglandin E2 (PGE2) and PGF2 alpha from the time of grafting, which was accompanied by a rise in the levels of prostacyclin (PGI2, measured as its stable hydrolysis product 6-oxo-PGF1 alpha) and thromboxane A2 (TxA2, measured as its stable hydrolysis product TxB2). When the level of each individual COP in rejecting graft tissue was expressed as a percentage of the total COP measured, PGI2 and TxA2 increased from 9% to 36% and from 7% to 25%, respectively, from day three after grafting until the time of graft rejection. In contrast to these changes in COP levels in rejecting grafts, the levels of all four COP in nonrejecting allografts maintained by treatment of the graft recipients with cyclosporine remained normal--i.e., comparable to the levels in the recipients' own hearts.

Use of monoclonal antibodies to quantitate T lymphocyte subpopulations in human cardiac allografts.

T cell subsets have been quantitated in 40 human cardiac biopsies to characterize the surface phenotype of T lymphocytes involved in acute rejection. Seventeen biopsies were from patients receiving conventional immunosuppression, and 18 were from patients receiving cyclosporine (Cys) as the major immunosuppressive drug. Five biopsies were from nontransplanted hearts. Frozen sections were treated with monoclonal antibodies of the Leu series and an immunoperoxidase technique to determine numbers of Leu 4 (T3 or pan-T-cell), Leu 2a (T8 or suppressor/cytotoxic), and Leu 3a (T4 or helper/inducer) positive cells. Biopsies from patients on conventional immunosuppression during rejection contained 68.3 +/- 10 infiltrating leukocytes/0.048 mm2 of the biopsy, of which 47 +/- 7.4% were T cells. Most of the T cells (86%) were of the 2a/T8 phenotype. In contrast, nonrejecting hearts contained substantially fewer infiltrating leukocytes (43.3 +/- 18.8/0.048 mm2), of which only 12.6% were T cells. There was no predominance of the 2a/T8 subset in nonrejecting biopsies. It is concluded that in patients receiving conventional immunosuppression, rejection is associated with an influx of T cells, most of which are 2a/T8-positive, into the cardiac allograft. In contrast, biopsies from patients on Cys contained large numbers of infiltrating leukocytes that did not appear to correlate with the histological assessment of rejection. However, in these patients, rejection was associated with an increase in the number of T cells with no consistent pattern of subset distribution. Biopsies from nontransplanted hearts contained few infiltrating leukocytes (31.5 +/- 9.4/0.048 mm2), and none of them were T cells.

The kinetics and specificity of lymphocyte infiltration of cardiac allografts in unmodified and cyclosporin-treated rats.

Lymphocyte infiltration of heart grafts has been monitored using pulses of Indium-111-labelled syngeneic lymphocytes. The cells were injected into cyclosporin (CSA)-treated or untreated rats that had received an allograft 1, 2, 4, 6, and 8 days previously Accumulation of the labelled cells in the graft was measured 24 hr after injection, and was compared with that in the animal's own heart. For the first three days after grafting, Indium-111-labelled lymphocytes accumulated to the same extent in the grafts of untreated and CSA-treated rats. However, the substantial rapid increase in lymphocyte accumulation in the graft that occurs in the untreated recipient between days 4 and 5 did not occur in CSA-treated recipients. From day 5 until day 9 the accumulation of labelled cells in CSA-maintained grafts was not greater than in syngeneic non-rejecting grafts, and it was significantly less than in the untreated rejecting grafts. At early times after grafting, removal from the labelled cell population of lymphocytes with reactivity against the histocompatibility antigens of the graft resulted in a reduction in the extent to which these lymphocytes accumulated in the graft.

A comparison of immune responses against AG-B and non-AG-B antigens, presented alone or together.

By exploiting congenic rat strains (HO.B2 and PVG/c) cell-mediated immune responses against Ag-B antigens alone were measured and compared with responses against (1) non-Ag-B antigens and (2) Ag-B and non-Ag-B antigens in combination. It was confirmed that multiple non-Ag-B antigens provoke prompt first-set skin graft rejections, but are much weaker than Ag-B antigens in stimulating both graft-versus-host (GVH) and cytotoxic activity. No evidence of synergistic interaction was found between anti-Ag-B and anti-non-Ag-B responses either by GVH assay or in the generation of cytotoxic cells. Specific partitioning of cytotoxic cells on antigenic monolayers suggested that cytotoxic cells on antigenic monolayers suggested that cytotoxicity is predominantly directed against Ag-B antigens. The measurements of GVH activity consolidate previous work, which suggested that 4.5 to 12% of nonimmune T cells can respond to each Ag-B determined antigenic complex and eliminate the possibility that most of these cells were responding to non-Ag-B antigens. Two principles for measuring GVH activity were compared: (1) 3H-thymidine incorporation into donor lymphocytes at 24 hr after transfer to irradiated F1 hybrid recipients and (2) the popliteal lymph node assay, which depends on a secondary phase of host cell proliferation.

Short course single agent therapy with an LFA-3-IgG1 fusion protein prolongs primate cardiac allograft survival.

The interaction of T cell costimulatory molecules with their ligands is required for optimal T cell activation. Interference with such interactions can induce antigen unresponsiveness and delay xeno- and allograft rejection. We have previously shown that LFA3TIP, a soluble human lymphocyte function-associated antigen (LFA)-3 construct, binds CD2 and inhibits responses of human T cells in vitro. This study reports the first use of a human fusion protein, LFA3TIP, to significantly prolong primate cardiac allograft survival. Based on our observations that LFA3TIP inhibits baboon allogeneic mixed lymphocyte reactions, we gave baboon recipients of heterotopic cardiac allografts injections of LFA3TIP, 3 mg/kg i.v., for 12 consecutive days, starting 2 days before transplantation. This regimen delayed graft rejection from an average of 10.6 +/- 2.3 days for human IgG-treated controls (n = 5) to an average of 18.0 +/- 5.3 days for LFA3TIP-injected animals (n = 7; P < or = 0.01). Grafts from LFA3TIP-treated animals showed markedly diminished coronary endothelialitis as compared with control animals. LFA3TIP reached peak serum levels of approximately 100 micrograms/ml after 7-9 injections and persisted in the 10-micrograms/ml range for 1 to 2 weeks after the final injection. Despite these blood levels, circulating antibodies to LFA3TIP were not detected in the serum. No renal or hepatic toxicity was noted. The possible mechanism by which LFA3TIP acts to inhibit graft rejection is discussed; success in prolonging graft survival when LFA3TIP is used as a single-agent therapy suggests great potential for this novel therapeutic agent.

Cell damage resulting from the labeling of rat lymphocytes and HeLa S3 cells with In-111 oxine.

Rat thoracic-duct lymphocytes and HeLa S3 cells were labeled in vitro with different amounts of indium-111 oxine. The labeled rat lymphocytes were tested for their ability to recirculate normally in syngeneic rats; the labeled HeLa S3 cells for their ability to divide to form colonies in tissue culture. Both cell types behaved normally by these criteria when labeled with small amounts of indium-111 oxine but at higher doses were obviously damaged. Evidence was obtained for the HeLa S3 cells that this damage was primarily radiation-induced. These findings may impose limitations on the use of In-111 oxine as a cell label for clinical purposes.

In vivo versus in vitro activity of cyclosporine.

Cyclosporine (CS) is a relatively new immunosuppressive agent which is selective in its action in that it acts on lymphocytes, T and B cells, and only indirectly if at all on other haemopoietic cells. Numerous studies over the last decade have shown quite clearly that, in vitro, CS inhibits lymphocyte activation at a relatively early stage by preventing the production and release of lymphokines, probably by inhibition of lymphokine gene transcription. However, studies in vivo have produced clearly different findings--although CS in vivo is completely effective in preventing the development of mature effector function (i.e., T-dependent antibody synthesis by B cells or T cell-mediated cytotoxicity) and the consequences of that effector function (e.g., transplant rejection), there is clear evidence that lymphocytes can be activated to at least the stages of cell division and clonal expansion under cover of therapeutic levels of CS. The contradictions between in vitro and in vivo findings are discussed in terms of the possible mechanism of action of CS in vivo.

Targeting of lymphocytes with 111In-labelled monoclonal antibodies.

In this paper, we emphasize the rationale and work-up studies for using two radiolabelled anti-lymphocyte monoclonal antibodies for in vivo application as radiolabelling agents for T and B cells. In vitro experimental work involved radioimmunoassays on human lymphoid cell lines and anticoagulated whole blood with identification of relevant binding kinetics in terms of antibody internalization and elution. We tested also for the effect of the radiolabelled monoclonal antibodies on in vitro cell function defined as mitogen-induced proliferation in whole blood. As a final work-up in an animal model, the distribution of both unlabelled and 111In-labelled anti-Pan T cell monoclonal antibody was studied in the rat. Results from the in vitro experiments pointed to the possibility of using the described technique for specific lymphocyte radiolabelling. The in vivo application enabled us to identify optimal doses of antibody and radioactivity which showed agreement with the in vitro data.

Does cyclosporine act in vivo as it does in vitro?

Cyclosporine aborts the activation of lymphocytes in vitro before DNA synthesis begins, yet there is also compelling evidence that lymphocytes can become primed, i.e. presumably proliferate in vivo, under the cover of immunosuppressive levels of the drug. Here, Gerry Klaus and Patricia Chisholm discuss the possibility that cyclosporine has a different mode of action in vivo and in vitro.

Co-expression of CD4 and CD8 molecules and de novo expression of MHC class II antigens on activated rat T cells.

Rat T-cell populations were analysed for their surface expression of CD4, CD8 and class II MHC antigens using monoclonal antibodies (McAbs) W3/25, Ox8 and Ox6 in immunofluorescence and immunoenzyme cytochemistry. Resting T cells expressed either CD4 (W3/25) or CD8 (Ox8) molecules, but not both, and did not express class II MHC antigens. In contrast, when purified T cells were stimulated by Con A or by alloantigens in a mixed lymphocyte reaction (MLR), over 60% of the cells expressed class II MHC antigens after 48-72 hr. The expression of class II MHC antigens by the T-cell blasts was transient and no longer evident after 96 hr of activation. In addition, T cells activated by either alloantigen or lectin expressed CD4 (W3/25) and CD8 (Ox8) molecules simultaneously: 30% of cells activated in MLR and 50% of Con A-activated cells expressed both markers within 48 hr of activation. The overlapping of the CD4- and CD8-positive subpopulations within the T-cell population was inferred from the results of staining for each marker separately and confirmed using a double-immunoenzyme staining method.

Selection of antigen-specific cells by adherence to allogeneic cell monolayers: cytolytic activity, graft-vs.-host activity and numbers of adherent and nonadherent cells.

Rat lymph node cells taken at the peak of cytolytic activity following a skin allograft were separated into adherent and nonadherent fractions by incubation on monolayers of thoracic duct lymphocytes either of the same strain as the graft donor or of an Ag-B different strain. In the face of a 3-fold enrichment of cytolytic activity in the adherent cells and a 3-fold depletion in the nonadherent cells there was no detectable partition of graft-vs-host (GVH) activity. Supplementary experiments supported the simplest interpretation of this finding, namely that the antigen receptors on GVH-reactive cells did not influence their adherence in this system. Similarly, there was no partition of the GVH activity of nonimmune lymph node cells by adherence. Labeling lymph node cells with either radioactive uridine or thymidine in vitro, suggested that about 20% of DNA-synthesizing cells in the immune population adhered because of antigen recognition.

Monoclonal antibodies to the integrin alpha-4 subunit inhibit the murine contact hypersensitivity response.

Lymphocyte-endothelial cell recognition is an active multistep process central to the pathophysiology of inflammation. In vitro models of lymphocyte adhesion predict that the beta 1 integrin very late antigen-4 (VLA-4), an activation-dependent adhesion receptor, can mediate the firm sustained attachment required for the extravasation of memory lymphocytes. We have used murine contact hypersensitivity as an in vivo model in which to evaluate the role of alpha-4 integrins in an evolving inflammatory response. We demonstrate that the intravenous administration of 75 micrograms of the anti-alpha-4 specific monoclonal antibodies R1-2 or PS/2 4-6 h prior to challenge significantly inhibits the efferent response of 2,4 dinitrofluorobenzene, or oxazolone-sensitized mice. The disease-modifying effect of anti-alpha 4 treatment was evident as a 50-60% reduction in the ear swelling response. By histological analysis treated animals scored lower for edema, number of epidermal lesions and degree of leukocyte infiltration. Antibody-treated animals have elevated numbers of circulating mononuclear leukocytes present in the same relative ratio as untreated control animals, suggesting that the inhibitory effect was not due to antibody-dependent cellular depletion of effector lymphocytes. These data are consistent with a central role for VLA-4 in the pathophysiologic process of inflammation.

Experimental models of bacterial arthritis: a microbiologic and histopathologic characterization of the arthritis after the intraarticular injections of Neisseria gonorrhoeae, Staphylococcus aureus, group A streptococci, and Escherichia coli.

We report the first reproducible experimental model of gonococcal arthritis, utilizing the intraarticular injection of virulent strains of N. gonorrhoeae. An acute, purulent synovitis with a marked polymorphonuclear leukocyte infiltrate developed during the 1st 24 h and persisted for at least 72 h, followed by a more chronic, mononuclear, proliferative synovitis. The histopathologic characteristics of this acute, then chronic synovitis were comparable to that after the intraarticular injection of other bacteria. N. gonorrhoeae could not be recovered from the injected joints 24 h after their inoculation, whereas Staphylococcus aureus, hemolytic streptococci, and E. coli could be recovered for days to weeks after the intraarticular injection.

Ketoacidosis occurring in newly diagnosed and established diabetic children.

A 6-y retrospective case note review was performed to determine the causes of ketoacidosis. 135 patients and 463 diabetic years were involved. Fifty-two ketoacidosis episodes occurred: 19 episodes in new patients and 33 episodes in 19 patients with established diabetes. 27% of newly diagnosed patients presented in ketoacidosis. They were similar in terms of age, sex and proportion living in single parent families to those presenting without ketoacidosis. The 33 ketoacidosis episodes occurring in established patients included 12 episodes in 3 children who were transferred to our care because of uncontrolled diabetes. Insulin omission was the cause of ketoacidosis in 9/19 (47%) patients, and was suspected in a further 5/19 (26%). Family and school problems were common and 14/19 patients came from single parent families. Established patients aged > or = 11 y were predominantly female (10F, 2M), whereas patients aged < or = 10 y were predominantly male (6M, 1F). 7 patients with multiple ketoacidosis episodes were all > or = 11 y and 6 were female. Families with > or = 2 diabetic children appeared vulnerable, 4 cases coming from 3/7 such families.

In vivo imaging of rat lymphocytes with an indium 111-labelled anti-T cell monoclonal antibody: a comparison with indium 111-labelled lymphocytes.

An indium 111-labelled mouse anti-rat T cell monoclonal antibody, MRC OX-19, was injected intravenously into rats to establish the usefulness of radiolabelled anti-lymphocyte antibodies in imaging lymphoid tissues. Antibody binding in vivo, measured by immunofluorescence analysis of cell suspensions made from lymphoid tissues, was detectable on lymphocytes in blood, spleen and lymph nodes. The extent of binding was time and antibody-dose dependent. Doses of antibody above 80 micrograms/kg body weight resulted in modulation, i.e. loss of CD 5 (T1) molecules from the cell surface, although the cells remained in the circulation. Modulation was demonstrable within 2 h and for at least 24 h after a single injection of antibody. Intravenous injection of 111In-MRC OX-19 resulted in levels of in vivo binding comparable with those seen with unlabelled antibody. Scintillation imaging showed early splenic localisation persisting over 48 h, a more gradual localisation in the lymph nodes seen clearly at 24 h and a steady background. Comparison of the in vivo distribution of labelled antibody and 111In-tropolone-labelled lymphocytes showed that both could be used for external imaging of lymphocytes by scintillation camera.

The effects of cyclosporin on lymphocyte activation in a systemic graft-vs.-host reaction.

We have investigated the effects of cyclosporin (CsA) on each of three stages of lymphocyte activation in vivo viz. sequestration of alloantigen-reactive lymphocytes from the circulation into the spleen and lymph nodes, blast transformation and induction of DNA synthesis in the activated cells and release of these cells and their progeny into the circulation. Parental strain lymphocytes injected i.v. into semi-allogeneic rats and recovered from the thoracic duct within 36 h are profoundly unresponsive in a local graft-vs.-host assay to the alloantigens of the F1 hybrid but have normal activity against unrelated alloantigens (negative selection). CsA treatment of the F1 hybrid recipients did not prevent this selective sequestration of antigen-reactive cells. In the untreated F1 hybrid, from 36 h after injection, large numbers of dividing blast cells were released into the lymph. These cells did not appear in the lymph of recipients treated with CsA. However, CsA did not prevent the activation of cells sequestered in the spleen or lymph nodes as assessed by [3H] thymidine incorporation and autoradiography. This unexpected finding suggests that CsA inhibits lymphocyte responses to alloantigens in vivo after DNA synthesis which is a later stage than the in vitro studies have shown.