The role of experimental aluminum intoxication in allogeneic immunoresponse

To evaluate the immunological properties of aluminum (Al) in experimental Al intoxication in rats, we performed heart transplantation and in vitro experiments. Lewis (Lew) rats were intoxicated with intraperitoneal injections of AlCl₃. Heart transplants were performed using Brown-Norway (BN) rats as donors. Isotransplants and normal Lew were used as controls. No differences in survival were observed. Unidirectional mixed lymphocyte cultures (MLC) and Concanavalin A (Con A)-stimulated cultures were prepared using spleen cells from normal and Al-intoxicated Lew rats. No differences were found in unidirectional MLC. Intoxicated cells showed a less intense response to Con A than did normal cells. In conclusion, we could not detect an immunosuppressive role of Al intoxication in experimental cardiac transplantation or in MLC. However, the depressed Con A blastogenic response of Al-intoxicated cells may reflect an immunological role yet to be defined.     

A reappraisal of the effects of monoclonal antibodies directed at T cell differentiation antigens

We examined the effects of monoclonal antibodies (Abs) directed at T3 antigen (expressed on most post-thymic T cells), T4 antigen (helper/inducer subset) and T8 antigen (suppressor/cytotoxic subset). Anti-T3 induced interleukin-2 production and proliferation of peripheral blood mononuclear cells (PBM). Anti-T4 or T8 did not exhibit such properties. Addition of methylprednisolone (MP) or cyclosporine (CsA) to PBM activated with anti-T3 resulted in 79% and 88% suppression of proliferation, respectively. Neither anti-T4 nor anti-T8 mediated significant inhibition of anti-T3-induced proliferation. Primary mixed lymphocyte cultures (MLC) were variably affected by Abs. Anti-T3 augmented proliferation found in primary MLCs at 48 hr and had an inconsistent effect on the proliferative response found at 120-136 hr of culture. Primary cytotoxic T lymphocyte (CTL) generation was consistently suppressed by anti-T3, while natural killer (NK)-cell-like activity was augmented at 72 hr and suppressed after 136 hr of culture. Anti-T4 mediated a dose-dependent suppression of proliferation and CTL generation in the primary MLC and had minimal effect on the induction of NK-cell-like activity. At high concentrations (5000-1000 ng/ml), anti-T8 mediated modest inhibition of proliferation and of the induction of cytolytic activity. Alloimmune memory cells, generated in long-term primary MLCs, were activated by anti-T3 to exhibit specific secondary cytolytic activity and NK-cell-like activity in the absence of the original priming stimulus. Neither anti-T4 nor anti-T8, under identical experimental conditions, activated memory cells. When interrelated, our experimental findings indicated that: (1) the ultimate immunity elicited by anti-T3-T3 antigen interaction is critically dependent upon the immune potential of the cell assessed; (2) MP or CsA can inhibit PBM activation by anti-T3; and (3) anti-T4 might have a role as an immunosuppressant in renal graft recipients.     

The effects of cyclosporine and cyclosporine metabolites in experimental small intestinal transplantation

Cyclosporine metabolites (CM) were compared with cyclosporine for their in vitro and in vivo immunosuppressive, nephrotoxic, and hepatotoxic effects using (A) in vitro mixed lymphocyte induction of monocyte/macrophage procoagulant activity (PCA), an accurate marker of allograft rejection; (B) in vitro toxic effects on renal cells in culture; and (C) a unidirectional rejection model of rat small intestinal transplantation (SIT). CM were composed of OL1, OL17, OL18, and two additional peaks C and H, (peak C: mass = 1235, 15.3% of total CM, peak H: mass = 1205, 6.3% of total CM). In vitro, CM fully suppressed the one-way mixed lymphocyte culture-induced PCA from 52.5 +/- 8.2 mU/10(6) PBM to the basal level 22.3 +/- 6.6 mU/10(6) PBM (P less than 0.01), which was comparable to CsA (21.3 +/- 5.5 mU/10(6) PBM). Lewis rats that had received Lewis-Brown Norway F1 hybrid intestinal allografts when treated with CM, demonstrated near-normal histology with minimal signs of rejection as compared with the fulminant clinical and histological rejection observed in the control (untreated and Cremaphor/NaCl treated) animals. PCA was markedly elevated in the control animals, 278 +/- 172 (untreated) and 160 +/- 98 mU/10(6) PBM (Cremaphor/normal saline treated), whereas CsA-treated allogeneic transplants expressed only basal levels of PCA (14.0 +/- 4 mU/10(6) PBM) (P less than 0.01), associated with normal histology. CM-treated animals expressed PCA levels of 27.0 +/- 10 mU/10(6) PBM, which was significantly different from both control and CsA-treated animals (P less than 0.01). In contrast to CsA-treated animals, CM-treated allogeneic transplants demonstrated no apparent renal or hepatic toxicity, as measured by blood urea nitrogen (25.3 +/- 9.5 vs. 10.0 +/- 5.3 mg/dl), alkaline phosphatase (160.7 +/- 29.3 vs. 100.3 +/- 19.5 U/L), and aspartate transaminase (96.7 +/- 23.7 vs. 61.7 +/- 11.7 U/L) (P less than 0.01). Similarly, in contrast to CsA, CM had minimal or no toxicity in renal epithelial and mesangial cells in culture, as measured by minimal or no inhibition of DNA, RNA, and protein synthesis. These results suggest that CM have potent immunosuppressive properties with no apparent nephrotoxicity and hepatotoxicity in vitro and in vivo.     

Cell-mediated cytotoxic activity of spleen cells from patients with gastric carcinoma

The cell-mediated cytotoxic activities of cells from the spleens (SP cells) of patients with gastric carcinoma were assayed in comparison with the activities of peripheral blood mononuclear cells (PBM cells) from the same patients, and from patients with benign lesions. The natural killer cell (NK) activity of the SP cells and their capacity to generate allogeneic cytotoxicity in mixed lymphocyte culture (MLC) were very similar to those of the PBM cells. The cytotoxic activity of SP cells induced by alloactivation in MLC, however, was significantly higher than that of the PBM cells from the same patient as well as from patients with benign lesions. The production of interleukin 2 (IL 2) and the ability to induce cytotoxic cells after activation with IL 2 (LAK) were therefore examined. Both the ability to produce IL 2 and to generate LAK cells were shown to be significantly increased in SP cells when compared to PBM cells. These results indicate that the spleen may be a potential reservoir for the precursors of these activated killer cells in patients with gastric carcinoma. Furthermore, it may play an important role in the defence against tumors in these patients.     

In vitro effect of interleukin 2 on depression of cell-mediated immune response after surgery

Effect of exogenous interleukin 2 (IL 2) on the postoperative depression of cell-mediated immune response in vitro was studied in 8 patients with benign lesion and 11 patients with various carcinoma, undergoing major surgical procedures. When peripheral blood mononuclear cells (PBM) were obtained from patients 3 days after surgery, the proliferative response of PBM in mixed lymphocyte culture (MLC) was significantly decreased, as compared to that before operation. The depressed proliferative response was significantly increased and improved to the preoperative level, when exogenous IL 2 was added at a concentration of 50 per cent in culture. The defective generation of cytotoxic cells in MLC 3 days after operation was also significantly augmented and improved to the preoperative range by addition of 25 per cent IL 2 in culture. IL 2 produced minimal increase in these responses when PBM were obtained preoperatively. There was no significant difference between each value in these responses obtained from patients with benign lesion and carcinoma. These results show that PBM from patients who had undergone major surgery were responsive to exogenous IL 2. The postoperative depression of cell-mediated immune response may be reversible with exogenous IL 2.     

Cell-mediated cytotoxic activity of spleen cells from patients with gastric carcinoma

The cell-mediated cytotoxic activities of cells from the spleens (SP cells) of patients with gastric carcinoma were assayed in comparison with the activities of peripheral blood mononuclear cells (PBM cells) from the same patients, and from patients with benign lesions. The natural killer cell (NK) activity of the SP cells and their capacity to generate allogeneic cytotoxicity in mixed lymphocyte culture (MLC) were very similar to those of the PBM cells. The cytotoxic activity of SP cells induced by alloactivation in MLC, however, was significantly higher than that of the PBM cells from the same patient as well as from patients with benign lesions. The production of interleukin 2 (IL 2) and the ability to induce cytotoxic cells after activation with IL 2 (LAK) were therefore examined. Both the ability to produce IL 2 and to generate LAK cells were shown to be significantly increased in SP cells when compared to PBM cells. These results indicate that the spleen may be a potential reservoir for the precursors of these activated killer cells in patients with gastric carcinoma. Furthermore, it may play an important role in the defence against tumors in these patients.     

In vitro effect of interleukin 2 on depression of cell-mediated immune response after surgery

Effect of exogenous interleukin 2 (IL 2) on the postoperative depression of cell-mediated immune responsein vitro was studied in 8 patients with benign lesion and 11 patients with various carcinoma, undergoing major surgical procedures. When peripheral blood mononuclear cells (PBM) were obtained from patients 3 days after surgery, the proliferative response of PBM in mixed lymphocyte culture (MLC) was significantly decreased, as compared to that before operation. The depressed proliferative response was significantly increased and improved to the preoperative level, when exogenous IL 2 was added at a concentration of 50 per cent in culture. The defective generation of cytotoxic cells in MLC 3 days after operation was also significantly augmented and improved to the preoperative range by addition of 25 per cent IL 2 in culture. IL 2 produced minimal increase in these responses when PBM were obtained preoperatively. There was no significant difference between each value in these responses obtained from patients with benign lesion and carcinoma. These results show that PBM from patients who had undergone major surgery were responsive to exogenous IL 2. The postoperative depression of cell-mediated immune response may be reversible with exogenous IL 2.     

Rat-SCID chimeras: Functional characteristics of the immune system of SCID mice after transplantation of rat fetal hematopoietic cells

Abstract: It has been demonstrated that allogeneic and xenogeneic lymphocytes can survive and expand in severe combined immunodeficiency (SCID) mice, but the T-cell function of the chimeras has not always been tested. The aim of this study was to develop a rat-SCID xenogeneic chimera and to examine the T cell response against RT1-incompatible rat cells. Fetal liver cells (FLC) from Lewis (LEW) rats were injected i.v. into irradiated C.B-17-scid (SCID) mice at a dose of 4×10⁷ cells/mouse. After 4 weeks, FACS analysis of peripheral blood lymphocytes (PBL) with monoclonal antibodies specific for rat lymphocyte subpopulations showed full reconstitution. The mice carried 6.2(±1.6)×10⁶ PBL/ml and 92.5 (±39.5)×10⁶ cells in the spleen. PBL, spleen, and lymph nodes showed distribution of CD4, CD8, and RT1 rat cellular markers similar to that observed in normal rats. Spleen cells from rat-SCID chimeras demonstrated normal proliferative T cell responses to Concanavalin A. Chimeras were primed i.p. with 10⁷ Brown Norway (BN) and ACI rat spleen cells, and the responder spleen cells of these primed chimeras showed specific proliferative responses in mixed lymphocyte culture (MLC) against BN and ACI stimulator lymphoid cells, respectively. Specific proliferative responses of rat-SCID chimeras were higher against ACI than BN stimulators. These MLCs also expanded cytolytic T lymphocytes that lysed ⁵¹Cr labeled BN and ACI targets at levels up to 50–90%, respectively. In conclusion, rat FLC injected into SCID mice differentiate and repopulate the immune system as well as function in generating alloantigen-specific cytolytic and helper T cells.     

Platelet-activating factor primes endotoxin-stimulated macrophage procoagulant activity

Macrophage procoagulant activity (PCA) at the site of inflammation may be induced by several stimuli including bacteria and endotoxin (LPS). The local factors controlling PCA induction are poorly defined. The lipid mediator platelet-activating factor (PAF) is ubiquitous to inflammatory sites. To determine the effect of PAF on LPS-induced PCA, thioglycolate-elicited murine peritoneal macrophages were exposed to PAF (10(-7) M) or control medium for 30 min and then stimulated with LPS (10 micrograms/ml) for 2, 4, or 6 hr. The ability of macrophages to shorten the clotting time of plasma (ie., PCA) was then measured and clotting times were converted to PCA units using a thromboplastin standard. Cytosolic calcium ([Ca2+]i) measurements were made using the calcium-sensitive fluorescent dye indo-1. PAF alone did not induce a rise in PCA expression (medium alone, 47 +/- 11 mU/10(6) cells; PAF alone, 49 +/- 12 mU/10(6) cells at t = 4 hr), but PAF treatment prior to LPS exposure resulted in a significant increase in the LPS-stimulated expression of PCA (LPS alone, 190 +/- 29 mU/10(6) cells; PAF/LPS, 329 +/- 57 mU/10(6) cells at t = 4 hr, P less than 0.05). This priming effect was reversed by the PAF antagonist WEB 2086 (WEB/PAF/LPS, 196 +/- 31 mU/2 x 10(6) cells). Stimulation of cells with PAF alone resulted in a rapid rise in [Ca2+]i (resting, 213 +/- 19 nmole; peak, 577 +/- 35 nmole). This effect was also inhibited by WEB 2086. These data suggest that PAF plays an important role in the modulation of PCA production by macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immune responses in small intestinal transplantation in the rat: correlation of histopathology and monocyte procoagulant activity

No predictive serologic marker exists for rejection or graft versus host disease (GVHD) reactions in small intestinal transplantation (SIT). SIT was performed in Lewis (Lew) and Lew X Brown Norway Fl hybrid (LBN) rats in the following combinations: group 1, Lew X Lew; group 2 (isolated rejection), LBN X Lew, and group 3 (isolated GVHD), Lew X LBN. Procoagulant activity (PCA), an index of monocyte immune activation, was measured in the peripheral blood mononuclear cells of graft recipients to assess its value as an immunologic monitor. Histologic findings and PCA were evaluated on days 1, 2, and 3 and every 2 to 3 days after SIT. No pathologic findings of graft or host tissue developed in group 1 (n = 14). Histologic rejection (blunted villi and mononuclear cell infiltration) was seen beginning on day 5 in group 2 (n = 19); early GVHD (loss of nodal and splenic architecture) was first noted on days 5 and 6 in group 3 (n = 17). PCA elevation in SIT was seen to precede histologic evidence of rejection or graft versus host disease in this model and may constitute an important marker for these immunologic events.     

Synergistic effect of donor-specific blood transfusion and a short course of deoxyspergualin in rat kidney transplantation

Deoxyspergualin (DSG), an analogue of spergualin produced by B. laterosporus, has a strong immunosuppressive effect in various transplantation models. We have investigated the mechanism of donor-specific prolongation of survival time in rat kidney grafting by donor-specific blood transfusion (DST) and a short course of DSG. Lewis (LEW) kidney allografts were transplanted into fully allogeneic BN rats. Fresh, whole LEW blood 1.0 ml, was injected i.v. into BN rats 2 days prior to transplantation. Then, DSG, 6 mg/kg per day, was administered by i.m. injection on days 0, 1, and 2 after transplantation. The recipients were divided into five groups: group 1 (n=6) no treatment: group 2 (n=6) DST only; group 3 (n=7) DSG only; group 4 (n=7) DST and DSG; and group 5 (n=6), third party (ACI rats) blood transfusion and DSG. Lymphocytes (cervical lymph nodes) and serum were harvested from BN recipients on day 7 postgrafting. For suppressor cell assays, lymphocytes from BN recipients in each group were added as a third cell to the mixed lymphocyte reaction (MLC) between nontransplanted BN lymphocytes (responder) and LEW or other third party (PVGC, ACI, WKA rats) lymphocytes (stimulator). Antidonor lymphocytotoxic antibody (ADLA) was checked by microcytotoxicity assays. Median survival times (MST) for each group were: group 1, 10 days; group 1, 10 days; group 3, 13 days; group 4, 75 days; and group 5, 13 days. Remarkable prolongation of MST was only noted in group 4. In the suppressor cell assay, group 4 showed significant suppression (40%; P<0.05); the other groups did not show any suppression. This suppressive activity in group 4 was effective only during the MLC between BN and LEW, not during the MLC of third party-BN combinations. Thus, suppressor cells from DST/DSG-treated BN recipients appear to be donor-specific. In the microcytotoxicity assay, the only group that showed any ADLA was group 2, which was not treated with DSG. These results clearly show that both induction of donor-specific suppressor cells and inhibition of ADLA production are associated with the remarkable donor-specific prolongation of kidney allograft survival in DST/DSG-treated recipients.     

Immunoregulation and apoptosis induction of nitric oxide in the human mixed lymphocytes culture

Nitric oxide (NO) is a multifunctional molecule in a variety of physiologic and pathologic processes. Its precise effect on human T lymphocyte responses against alloantigens are not yet fully known, although it has been reported that NO is antiproliferative and can cause apoptosis in several cell types. To address these issues, we analyzed the effects of an NO donor on mixed lymphocyte cultures (MLC) and on apoptosis induction in T lymphocytes activated with alloantigens. NOC 18 was used as an NO donor. The MLC was performed with human peripheral blood mononuclear cells isolated from healthy volunteers. Cell division and interleukin (IL)-2 production were measured with CFSE labeling and an EIA kit, respectively. After cells were incubated with NOC 18 for 24 hours, DNA fragmentation was assessed using the diphenylamine assay. Pre-culture of cells with NOC 18 for 24 hours resulted in significant inhibition of cell proliferation and IL-2 production in MLC. NOC 18 induced DNA fragmentation of cells harvested from an MLC following 7 days of the culture, in a dose-dependent manner, whereas it never exerted any influence on DNA fragmentation of freshly isolated cells. A chemical NO donor, NOC-18, may have immunosuppressive ability when treatment of responder cells occurs before the beginning of the MLC and may induce apoptosis of alloantigen-activated T lymphocytes.     

The effects of perioperative portal venous inoculation with donor lymphocytes on renal allograft survival in the rat. I. Specific prolongation of donor grafts and suppressor factor in the serum

In order to investigate the in vivo functional role of the liver in the immune responses in organ transplantation, effects of perioperative portal venous p.v. administration of donor lymphocytes on renal allograft survival were tested in the rat kidney transplant model. Donor lymphocytes were prepared from BN (BN, RT-1n) or third-party DA (RT1a) rat spleens and lymph nodes and injected p.v. or intravenously to Lewis (LEW, RT-1l) hosts on the day of transplantation (day 0). Untreated LEW hosts rejected BN renal grafts at 7.8 +/- 0.6 days (n = 10). Intravenous administration of 1 x 10(8) BN cells to LEW hosts on day 0 caused a slight, but not significant, prolongation of renal allograft survival (MST = 9.5 +/- 3.0 days, n = 13, NS), whereas portal venous inoculation of 1 x 10(8) BN cells on day 0 remarkably prolonged renal graft survival to 22.2 +/- 5.3 (n = 10, P less than 0.01). The prolongation of graft survival was antigen-specific; the administration of 1 x 10(8) DA cells p.v. to LEW hosts did not prolong the survival of BN renal grafts (MST = 7.4 +/- 0.8, n = 5). Spleen cells from p.v. treated LEW hosts 10 days after transplantation had no suppressor effect on the one-way MLC reaction of normal LEW responder cells toward donor BN or third-party DA stimulators. On the other hand, when serum from p.v.-treated LEW hosts was added to MLC at a concentration of 3 per cent of total volume, it suppressed the MLC reaction toward donor BN cells by 71.6 per cent, but not toward third-party DA stimulators (-8.5 per cent suppression, NS). Histological examination of p.v.-treated LEW hosts at 10 days after transplantation revealed that the liver had normal lobular architecture without expansion of portal tracts and infiltration of inflammatory cells. On the other hand, the transplanted kidney demonstrated a moderate mononuclear cell infiltration around the artery without an interstitial hemorrhage. Moreover, adoptive transfer of the serum from p.v.-treated LEW rats into the virgin secondary LEW hosts significantly prolonged the graft survival of BN kidneys from 7.8 days to 18.9 +/- 5.5 days (P less than 0.01), but not third-party DA graft survivals (MST = 7.5 +/- 0.6 days), indicating that an antigen-specific tolerogenic factor was released into the circulation through the process of allogeneic cells in the liver.     

Detection of alloimmune memory cells by chemical modification of the cell surface with mitogenic oxidizing agents

The mitogenic oxidizing agents, neuraminidase and galactose oxidase (NAGO), and sodium periodate (IO-4) were used to induce the differentiation of human alloimmune memory cells. NAGO or IO-4 treatment of peripheral blood mononuclear (PBM) cells obtained from 10 sensitized potential allograft recipients resulted in the induction and augmentation of cytolytic activity to a D locus-defined lymphoblastoid cell panel (B cell panel) and to a HLA-disparate peripheral blood lymphocyte cell panel (PBL cell panel). The acquisition of cytolytic activity was determined in a 4-hr 51Cr release assay. Treatment of in vitro-primed PBM cells (alloimmune memory cells generated in primary long-term mixed lymphocyte cultures) obtained from normal subjects with NAGO or IO-4 also resulted in the induction of specific secondary cytolytic activity. In contrast, NAGO or IO-4 treatment of unprimed PBM cells from normal subjects did not result in the induction of cytolytic activity despite the extensive proliferation induced by such treatment. The strikingly similar results observed with PBM cells from sensitized patients and with in vitro-primed PBM cells suggest that in vivo- and in vitro-generated alloimmune memory cells can be detected by chemical modification of the cell surface induced by NAGO or IO-4. Furthermore, our findings indicate that alloantigen-independent activation of memory cells can be accomplished by treating the memory cells with the mitogenic oxidizing agents.     

Graft-versus-host disease in fully allogeneic small bowel transplantation in the rat

Small bowel and its mesentery contain considerable amounts of lymphoid tissue that can mediate graft-versus-host disease in small bowel transplant (SBT) recipients. Present studies determined the existence of GVHD in a fully allogeneic SBT model and examined the effect of donor pretreatment with ALS in eliminating GVHD. Adult male Lewis (Lew) rats received orthotopic small bowel transplants from untreated (LewxBN)F1 (LBNF1) donors (group 1) or Brown Norway (BN) donors that were untreated (group 2) or pretreated with ALS (days -2 and -1) (group 3). All recipients were treated with cyclosporine 15 mg/kg/day i.m. on days 0-6 postoperatively. Animals were weighed and examined daily for signs of rejection and GVHD. No animals in groups 1 or 3 showed any physical signs of GVHD, but all of those in group 2 had characteristic weight loss, diarrhea, and dermatitis between 4 and 6 weeks postoperatively, from which they all recovered. Histologic examination of skin and spleen at this time confirmed the presence of GVHD. The relative spleen weight [( spleen weight/body weight] x 100) of group 2 animals was also significantly greater than that of unoperated control Lew animals. Spleen cells obtained from group 2 animals at the time of subclinical GVHD, but not cells from group 1 or 3 animals, caused enlargement of popliteal lymph nodes when they were injected into the footpads of Lew rats. This study shows that GVHD can manifest itself in recipients of a fully allogeneic small bowel transplant even when rejection is prevented by effective immunosuppression with CsA. However, combined use of recipient treatment with CsA and pretreatment of donor animals with ALS eliminates all manifestations of GVHD.     

Is macrophage-induced microthrombosis during endotoxemia dependent on prostaglandin synthesis?

Steroids and nonsteroidal anti-inflammatory drugs (NSAID), in addition to inhibiting prostaglandin synthesis, have recently been shown to block production of procoagulant activity (PCA) by alveolar macrophages (A-M phi). This inhibition may ameliorate one deleterious effect of endotoxin (LPS), since LPS-induced PCA may contribute to the diffuse microvascular thrombosis (DIC) and subsequent organ failure seen in endotoxemia. However, the results of treatment of endotoxemia with steroids of NSAID are variable or short-lived. To help elucidate the basis for these results, the effect of these agents on production of PCA by another M phi population, the hepatic M phi (H-M phi PCA is functionally discrete from A-M phi PCA) was evaluated. Four agents, (hydrocortisone, indomethacin, ibuprofen and acetylsalicylic acid (ASA)) used to both block production of prostaglandins and ameliorate the deleterious effects of LPS in vivo were tested on the production of rabbit M phi PCA in vitro in homogeneous cultures. PCA was measured by a standardized one-step coagulation assay. Treatment with LPS (1 to 10 g/ml) produced maximal stimulation of PCA production with a peak at 8 hr with PCA levels 20- to 30+-fold higher than controls. The effect of pretreatment (T-10 min) of M phi cultures with the various inhibitors on PCA production at 8 hr post-LPS was evaluated, (N = 6 to 10 experiments).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that stimulator cell-derived IL-6 and IL-1 are released in the mixed lymphocyte culture but are not requisite for responder T cell proliferation.

We investigated whether IL-6 and (or) IL-1 are crucial costimulatory signals in the human MLC with purified responder T cells. With allogeneic PBMC as stimulators, IL-6 and IL-1 were rapidly produced, and reached plateau values of 100-300 U/ml and 200-500 pg/ml after 24 hr, respectively. Irradiated or mitomycin-c treated PBMC could easily be induced (with LPS) to produce IL-6 and IL-1 while no activity was measured after 48 hr in the supernatant of PHA-stimulated T cells, suggesting that in the MLC the monokines were entirely produced by stimulator PBMC. In cultures of responder T cells and stimulator B cells, no IL-6 and IL-1 activity was measured in the supernatant, and only a marginal proliferative response was found. Exogenous IL-6 and IL-1 increased in a dose-dependent way the B-cell-induced alloresponse and induced significant cytotoxicity in the responder cells. Antisera to IL-6 and IL-1 totally inhibited the induced response. The proliferation was accompanied by increased IL-2 production and IL-2R expression. Preincubation of B cells with IL-6 and IL-1 did not improve the proliferation, suggesting direct effects of IL-6 and IL-1 on the T cells. The proliferative responses induced by B cells and exogenous IL-6 and IL-1 represented a fraction of those induced by PBMC. Moreover, in PBMC-stimulated cultures exogenous IL-6 and IL-1 or antisera to these lymphokines did not significantly alter proliferative responses, cytotoxicity, IL-2 levels in the supernatant, or IL-2R expression on responder T cells. We conclude that a role for IL-6 and IL-1 in allogeneic T cell stimulation can be demonstrated in conditions of suboptimal stimulation with B cells. With PBMC, neutralizing antisera to these cytokines do not seem to inhibit the proliferative response, suggesting that these cells are superior in alloantigen presentation either by producing various costimulatory signals or by the fact that due to cell-cell contact stimulator cell-derived monokines cannot be blocked. This finding makes it unlikely that antimonokine therapy will be useful in transplantation.     

Prolongation of rat kidney allografts by pretransplant administration of donor antigen extract or whole blood transfusion combined with a short course of cyclosporine

The immunosuppressive effect of the combination of a three day course of cyclosporine with one i.v. injection of 3M KCL-extracted donor antigen or donor blood transfusion was tested across the strong histocompatibility barrier causing rejection within 8 days of kidney transplants from Buffalo (Buf, RT1b) to Wistar-Furth (WFu, RT1u) inbred rats. Administration of 10 mg/kg/day cyclosporine alone for three days (-1, 0, and 1) slightly prolonged graft survival time from 7 to 11 days. The combination of cyclosporine with donor Buf 3M KCl antigen or with a Buf blood transfurion administered one day prior to transplantation caused greater prolongation of graft survival--19 and 25 days, respectively. Neither third-party BN soluble antigen nor BN blood transfustions acted synergistically with cyclosporine to prolong Buf graft survival. Increasing doses of donor-soluble antigen up to an optimal dose of 5 mg proportionately prolonged graft survival; however, administration of 10 mg antigen was less effective than 5 mg. On the other hand, administration of 1 ml of donor blood achieved the maximal effect. Lymphocytes harvested ten days after transplantation from recipients that had received combined therapy with cyclosporine and donor 3M KCl antigen not only displayed specific unresponsiveness to donor stimulator cells in mixed lymphocyte culture, but also specifically suppressed the proliferative response of syngeneic, virgin WFu responder cells to allogeneic donor Buf but not to third-party BN cells. Furthermore, suppressor cell activity was suggested by diminished responses in an in vivo local adoptive mixed lymphocyte culture assay and by prolongation of Buf kidney survival following systemic adoptive transfer. These findings suggest that immunosuppression with cyclosporine permits induction of specific suppressor cells by 3M KCl donor antigen, resulting in specific unresponsiveness to allografts.     

Fibronectin restores defective in vitro proliferation of patients' lymphocytes after marrow grafting

In search for means of improving the impaired lymphocyte function of recipients after marrow grafting, we investigated the effect of fibronectin (FN) on patients' lymphocytes in allogeneic mixed lymphocyte cultures (MLC) and in cell-mediated lympholysis (CML) assays, since these tests are usually defective in transplanted patients. Four subgroups of marrow recipients were tested: patients within the first 100 days of transplantation (short-term) with (n = 16) or without (n = 14) acute graft-versus-host disease (GVHD), and long-term recipients with (n = 23) or without (n = 15) chronic GVHD. Exogenous FN (25 micrograms/ml) increased the proliferative response in the allogeneic mixed lymphocyte culture (MLC) significantly in cells from short-term patients without acute GVHD (+42%) and in those from long-term recipients with (+117%) and without chronic GVHD (+48%). In cells from patients with chronic GVHD, 3H-thymidine uptake after the addition of FN was enhanced to the level of that in lymphocytes of the corresponding marrow donor without exogenous FN. Fibronectin was effective only if added at the beginning of the MLC. In contrast to the results in MLC, exogenous FN failed to enhance phytohemagglutinin or OKT-3-induced lymphocyte proliferation and had no effect on CML activity. Moreover, FN did not show mitogenic activity in 3-6-day cultures. Our results demonstrate that FN in vitro is capable of restoring defective lymphocyte proliferation in marrow grafted patients, and circumstantial evidence suggests that this effect is mediated by an interaction between FN and monocytes.     

白细胞介素4在诱导新生期大鼠免疫耐受中的作用

目的　探讨白细胞介素４（ＩＬ４） 在诱导新生期大鼠免疫耐受中的作用。方法　将２ ．５ ×１０７ 个Ｗｉｓｔａｒ 大鼠脾淋巴细胞注入新生ＳＤ 大鼠胸腺内，４～６ 周龄时取其脾淋巴细胞与Ｗｉｓｔａｒ 大鼠脾淋巴细胞（２０ Ｇｙ Ｘ 射线照射） 进行混合培养， 于７２ ｈ 测定ＩＬ４ 和γ干扰素（ＩＦＮγ） 的含量。结果　实验组ＩＬ４ 和ＩＦＮγ的含量分别为（４５５±８５） ｎｇ／Ｌ和（１４５±４６） ｎｇ／Ｌ； 对照组则分别为（１３９ ．２ ±４６ ．３） ｎｇ／Ｌ和（４７０±１０２） ｎｇ／Ｌ， 两组比较，ＩＬ４ 和ＩＦＮγ含量的差异均有显著性（ Ｐ＜ ０．００１） 。结论　ＩＬ４ 在新生期大鼠免疫耐受的诱导中起重要作用。