The selectivity of isoprinosine, NPT 15392, avridine and cyclophosphamide on multiple immune responses in rats

Multiple concomitant immune responses were assessed in individual rats following treatment with the immunoenhancing drugs, isoprinosine (5 or 50 mg/kg), NPT 15392 (0.1 or 1.0 mg/kg) and avridine (1 or 25 mg/kg), or the immunosuppressant, cyclophosphamide (75 mg/kg). Immune responses assessed in each rat were specific antibody synthesis, delayed-type hypersensitivity (DTH), natural killer cell (NKC) cytotoxicity and production of three immunoregulatory cytokines, interleukin 1 (IL1), interleukin 2 (IL2) and prostaglandin E2 (PGE2). Spleen and thymus weights and numbers of splenocytes and resident peritoneal cells were also recorded. Rats treated with isoprinosine had dose-related, significant increases in spleen weights and DTH reactions. Rats treated with NPT 15392 had significantly enhanced DTH reactions at the 0.1 mg/kg dose. Rats treated with the 25 mg/kg dose of avridine had significantly increased spleen weights, DTH reactions and NKC cytotoxicity. The effect of avridine treatment on DTH reactions and IL1 and IL2 production was inverse to the dose administered, while the NKC response was directly related to the dose. Thymus weights, antibody production and PGE2 synthesis were not significantly altered in rats treated with isoprinosine, NPT 15392 or avridine. Cyclophosphamide-treated rats had significantly reduced spleen and thymus weights, antibody synthesis, DTH reactions, NKC cytotoxicity and IL2 production, but IL1 and PGE2 synthesis were significantly elevated. It can be concluded that isoprinosine, NPT 15392 and avridine act as general immunostimulants in the rat, with avridine having the greatest effect under these experimental conditions. It also appears that these drugs are differentially immunoselective in the rat and this effect is at least partially related to the dose administered. These results could be of significance in the selective therapeutic manipulation of different arms of the immune system. Also, enhanced production of PGE2 following cyclophosphamide treatment may contribute to the immunosuppressive effects of this drug.     

Effects of NPT 15392 in vitro on human leukocyte functions

NPT 15392 (Erythro-9 (2-hydroxy-3 nonyl) hypoxanthine), a novel heterocyclic immunomodulatory compound, was analyzed over a broad concentration range on a variety of human blood leukocyte functions in vitro. NPT 15392 augmented mitogen-induced lymphocyte transformation in a variable fashion; lymphocytes from 9 of 24 individuals showed significant stimulation with phytohemagglutinin at 0.01 microgram/ml of NPT 15392, and 3 of 14 and 3 of 3 showed similar augmentation with concanavalin A and pokeweed mitogen, respectively. NPT 15392 above 10 microgram/ml inhibited mitogen responses and did not itself stimulate cell division. NPT 15392 also augmented responses of lymphocytes to antigenic stimulation with Candida and Staphylococcus antigens, purified protein derivative, and allogeneic cells in a variable manner. When observed, stimulation occurred at 0.01-1 microgram/ml of NPT 15392 for Candida and Staph. and at 0.01 microgram/ml with PPD and allogeneic cells. NPT 15392 (0.01-1 microgram/ml) consistently induced suppressor cell function alone and in combination with concanavalin A. This effect is apparently mediated by T lymphocytes since suppression was not mediated by interferon, prostaglandin or histamine. In addition, NPT 15392 (0.01-10 microgram/ml) significantly augmented "active" T cell rosettes. NPT 15392 over a broad concentration range and in the presence and absence of interferon did not stimulate natural killer cell activity or antibody-dependent cellular cytotoxicity. The data indicate that NPT 15392 is a modulator of such T lymphocyte functions as proliferative response to antigen and mitogen, suppressor activity and receptor display. Such activities imply potential therapeutic use in immunodeficiency related to defects of the thymus and thymus-derived lymphocytes.     

Effect of melatonin on lymphocyte proliferation and production of interleukin-2 (IL-2) and interleukin-1 beta (IL-1 beta) in mice splenocytes

The influence of Melatonin (MLT) on the modulation of the immune system has been described. In previous studies an increment of cell proliferation and an increase or a decrease of cytokines have been reported. Other workers have found inhibitory effects or no effect in the immune functions. Because of this controversy, and for the purpose of studying the mechanism by which MLT performs its functions, we evaluated its effect on murine splenocytes's proliferation after a mitogenic stimulation, and quantified the levels of IL-2 and IL-1 beta in the absence or presence of Phitohemaglutinin (PHA) in supernatants of mice splenocytes cell culture treated or not with MLT. The lymphoproliferative response was assessed using tritiated thimidine in the splenocytes of mice treated with 500 micrograms of MLT/Kg b.w. and in cell cultures containing 5, 50 and 100 micrograms MLT/mL. The production of IL-2 and IL-1 beta was detected by the ELISA test. An increase in the proliferation (p < 0.01) of spleen cells treated with 50 and 100 micrograms MLT/mL an optimal dose of PHA, was detected. The in vivo or in vitro treatment with MLT increased the levels of IL-2 and IL-1 beta in the absence or the presence of PHA, maintaining the increase in the concentration of IL-1 beta up to the to ninth day of treatment. These results suggest that MLT acts directly on cell proliferation probably by binding to high affinity receptors located on spleen cells, that stimulates the production of IL-2 and IL-1 beta giving rise to an increment of cell immunity.     

Effect of isoprinosine on sialylation of interleukin-2

The effects of Isoprinosine (ISO) on interleukin-2 (IL-2) production by human peripheral blood mononuclear cells (PBMC) were investigated. Treatment (of human PBMC) with ISO enhanced IL-2 production by PBMC from 7 of 10 normal individuals. However, no augmentation of IL-2 production was observed when cultures of HUT-78 cells, a human leukemic T cell line, were treated with ISO. IL-2 purified from supernatants of human PBMC treated with ISO exhibited pI values of 5.5 and 6.4. IL-2 prepared from untreated PBMC exhibited a single pI value of 8.2. The pI value of IL-2 prepared from ISO-treated PBMC shifted to 8.2 after treatment with neuraminidase, demonstrating that the IL-2 molecules isolated from ISO-treated PBMC possessed sialic acid. The pI values of the IL-2 isolated from ISO-treated and untreated HUT-78 culture supernatants were identical (pI = 7.8) and were not modified by neuraminidase treatment. These results suggest that the increase in IL-2 production following treatment of PBMC with ISO may be mediated through the activation of a distinct subset of IL-2 producing cells. Furthermore, the sialylation of IL-2 may be of physiologic and immunopharmacologic importance.     

Effect of isoprinosine on IL-2, IFN-gamma and IL-4 production in vivo and in vitro.

The effects of an immunopotentiating drug, isoprinosine, on the splenocytes of BALB/c mice to produce cytokines were investigated. Isoprinosine enhanced IL-2 production, upregulating the expression of IL-2 receptor in vitro. It also significantly increased the IFN-gamma secretion and decreased the IL-4 production in vivo. The significance of these findings in terms of immune regulation is discussed.     

Effects of moderate endurance exercise and training on in vitro lymphocyte proliferation,interleukin-2 (IL-2) production,and IL-2 receptor expression

This study was designed to examine immunological responses to an acute bout of cycle ergometry exercise before and after moderate endurance training. Previously sedentary males were randomly assigned to matched training (n=9) or control (n=6) groups. Training comprised 12 weeks during which supervised cycle ergometer exercise took place [30 min at 65–70% of maximal oxygen intake , 4–5 days · week^–1]. An acute bout of exercise (60 min; 60% was performed initially and after the 12-week interval. Samples of peripheral venous blood were taken at rest, after 30 and 60 min of exercise, and at 30 and 120 min post-exercise. Training improved by an average of 20% (40.6 to 49.2 ml · kg^–1 · min^–1). Relative to baseline and control measures, the resting concentration of (CD3-CD16⁺/CD56⁺) natural killer (NK) cells increased by 22% (P<0.05). The resting count of total CD25⁺ [interleukin-2 receptor (IL-2R) chain] lymphocytes did not change following training, but dual staining analysis showed a 100% increase in the fraction of CD16⁺ CD25⁺ NK cells (P < 0.05). Likewise the resting CD122⁺ (IL-2R chain) lymphocyte count increased 35% after training, the greatest increases (44%) being in CD16⁺ CD122⁺ NK cells (P<0.05). Soluble IL-2R levels also increased 33% (P< 0.05) after training. Following acute exercise at the same relative intensity; trained individuals exhibited a larger increase in the NK cell count, reduced lymphocytopenia, and attenuation of exercise-induced suppression of lymphocyte proliferation and IL-2 production (P<0.05). In addition, smaller increases in CD4 and CD8 counts during exercise were noted, but with faster recovery post-exercise (P<0.05). Addition of recombinant IL-2 (rIL-2) to phytohemagglutinin-stimulated peripheral blood mononuclear cell cultures did not reverse exercise-induced suppression of cell proliferation, either before or after training. However, rIL-2 did augment the spontaneous blastogenesis of exercise and post-training samples relative to baseline (P < 0.05). We conclude that moderate endurance training is associated with sustained alterations in immune function, both at rest and when exercising. Further investigations are necessary to determine the impact on overall health and susceptibility to disease.     

Kinetic studies of the immunopharmacologic effects of NPT 15392 in mice

NPT 15392 [9-erythro-(2-hydroxy,3-nonyl)-hypoxanthine] was administered in a single intraperitoneal injection to Balb/c mice at a dose of 0.1 mg/kg. Modifications of immune parameters were evaluated 1-14 days after the treatment. NPT 15392 potentiated antibody responses to both T-dependent (SRBC, TNP-KLH) and T-independent (TNP-LPS) antigens and delayed-type hypersensitivity to oxazolone. The proliferative response of spleen cells from NPT-treated mice to stimulation with PHA was depressed, but that to dextran sulphate was augmented. The responses to Con A or LPS were inconsistently modified. NPT 15392 augmented killer cell functions, including both T cell-mediated cytotoxicity against allogeneic tumor cells and NK cell activity against YAC-1 tumor cells. It slightly augmented or depressed ADCC activity against antibody-coated chicken erythrocytes (CRBC) depending on the time of its administration. Concerning the stimulation of NK cell activity, the effect was more marked on spleen effector cells when NPT 15392 was given i.v. and on peritoneal effector cells when it was given i.p. From these results, T helper cells, B cells, and NK cells appeared to be target cells of NPT 15392 action. The various stimulatory effects peaked at different times according to the immune function tested. In addition, the prolonged, sometimes double-peaked action (antibody response to T-dependent antigens, NK activity) indicates complex mechanisms of action which may involve indirect interactions mediated by lymphokines or monokines.     

Adoptive immunotherapy with interleukin-2 (IL-2) results in diminished IL-2 production by stimulated peripheral blood lymphocytes

We examined the responses of peripheral blood lymphocytes (PBL) to a panel of T-cell mitogens in patients receiving adoptive transfers of tumor-infiltrating lymphocytes and continuous infusions of interleukin-2 (IL-2) for treatment of advanced cancer. All patients showed diminished proliferative responses to soluble and alloantigens, lectins, anti-CD3, and IL-2 during therapy. The non-major histocompatibility complex (MHC)-restricted cytolytic activities of PBL were increased by treatment and were further augmented by IL-2in vitro. The expression of 55-kd low-affinity IL-2 receptors (IL-2R) by PBL increased during treatment but functional IL-2R were simultaneously down-regulated. Proliferative responses were partially restored to pretreatment levels when PBL were costimulated with recombinant IL-2 and mitogens. Lectin stimulation of PBL produced little IL-2 secretion during treatment, while IFN-gamma secretion persisted. We conclude that infusions of IL-2 down-regulate the expression of functional IL-2R, decrease the secretion of IL-2, and lead to decreased mitogen responses by PBL.     

Effects of interleukin-6 (IL-6) on cytotrophoblastic cells.

Tumour invasion and trophoblastic invasion share the same biochemical mediators: the matrix metalloproteinases (MMP) and their inhibitors. In contrast to tumour invasion of a host tissue, trophoblastic invasion during implantation and placentation is stringently controlled both in tissue localization and developmental stage. The factors responsible for these important regulatory processes are unknown, but in-vitro studies point to endometrial cytokines and growth factors as possible candidates. Here we examined the possibility that interleukin-6 (IL-6), a trophoblastic and endometrial cytokine, represents such a regulatory factor. Purified first trimester cytotrophoblastic cells (CTB) were cultured for 4 days in presence or absence of increasing concentrations of IL-6. MMP-2 and MMP-9 bioactivity (zymography) and immunoactivity were measured in the culture supernatants together with total human chorionic gonadotrophin (HCG), fetal fibronectin (FFN) and leptin. IL-6 did not change the cytotrophoblastic secretion of FFN or total HCG. In contrast, this cytokine induced a dose-dependent stimulation of the leptin secretion and increased the activity, but not the immunoreactivity, of MMP-9 and MMP-2. These results indicate that IL-6 could be considered as an endometrio-trophoblastic regulator of cytotrophoblastic gelatinases.     

In vitro effects of two immunopotentiators, Isoprinosine and NPT 15392, on murine T-cell differentiation and function

A new immunopotentiator, NPT 15392, was analyzed in vitro for its capacity to induce a T-cell marker (Thy-1 antigen) and to enhance the mitogen-induced lymphocyte proliferation. The activity was compared with that of Isoprinosine and a putative thymic hormone (thymosin Fr. V). Thymic hormone preparation (thymosin Fr. V) and the non-thymic agents (Isoprinosine and NPT 15392) induced significant percentages (20-30%) of Thy-1 positive cells in nylon wool-nonadherent nu/nu spleen cells. Maximum induction was observed the concentrations of 100 microgram/ml thymosin Fr. V, 1.0 microgram/ml Isoprinosine, and 0.1 microgram/ml NPT 15392. In addition, NPT 15392 or Isoprinosine significantly augmented proliferation of C57BL/6J spleen cells stimulated by the T-cell mitogens (Con A and PHA) and B-cell mitogen (LPS). The effect of both agents was greater on spleen cells from aged mice (18 mth) than on young mice (3 mth). The minimum effective concentration of NPT was lower than that of Isoprinosine. The data indicate that Isoprinosine and NPT 15392 have actions on precursor T cells, mature T cells, and probably B cells.     

Effects of immunization with tumor cells double transfected with interleukin-2 (IL-2) and interleukin-12 (IL-12) genes on artificial metastasis of colon26 cells in BALB/c mice

In order to induce tumor specific cytotoxicity, the poorly immunogenic murine colon cancer cell colon26 was transfected with murine IL-2 cDNA and/or IL-12 cDNA and their anti-tumor effects were investigated. Double transfectants produced murine IL-2 and murine IL-12, the same as single transfectants. Intraperitoneal administration of double transfectants inhibited pulmonary metastasis of colon26 inoculated intravenously to a stronger degree than that of single transfectants. Splenocytes from mice administered double transfectants intraperitonealy showed higher cytolytic activity against colon26 than those from mice administered single transfectants, and also showed cytolytic activity against murine B16-BL6 melanoma. In the NK cell-depleted mice, double transfectants inhibited pulmonary metastasis from the control markedly, but could not do completely, the same as in the NK cell-reserved mice. The difference of the metastatic colonies between NK cell-depleted mice and the control was much greater than that between NK cell-depleted mice and NK cell-reserved mice. These results suggested that cytotoxic T lymphocytes might participate in this anti-tumor effect.     

Lipoteichoic acid inhibits interleukin-2 (IL-2) function by direct binding to IL-2

Lipoteichoic acid (LTA) is associated with the cell envelope of most gram-positive bacteria. Although previously thought to act mainly as a virulence factor by virtue of its adhesive nature, evidence is now provided that LTA can also suppress the function of interleukin-2 (IL-2), an autocrine growth factor for T cells. LTA from four separate bacterial strains lowered the levels of detectable IL-2 during a peripheral blood mononuclear cell response to the antigen tetanus toxoid (TT). T-cell proliferation in response to TT was similarly inhibited by LTA. In contrast, levels of detectable gamma interferon increased. In addition, LTA inhibited IL-2 detection by enzyme-linked immunosorbent assay (ELISA) and blocked the proliferative response of an IL-2-dependent T-cell line to soluble IL-2. Further studies using ELISA demonstrated that LTA blocks IL-2 detection and function by binding directly to IL-2. Flow cytometric analysis revealed that IL-2 binding to T cells is inhibited in the presence of purified LTA but not LTA plus anti-LTA monoclonal antibody. In summary, these studies demonstrate a novel effect of LTA on the immune response through direct binding to IL-2 and inhibition of IL-2 function. Importantly, gram-positive organisms from which LTA is obtained not only play an important role in the pathology of diseases such as bacterial endocarditis, septic shock, acute respiratory distress syndrome, and multiple organ failure but also comprise a significant portion of commensal populations within the human host. Inhibition of IL-2 function by LTA may represent yet another mechanism by which gram-positive bacteria dampen the host immune response and facilitate survival. Thus, LTA provides a potential target for therapeutic intervention when gram-positive organisms are involved.     

In vivo effect of isoprinosine on interleukin-2 production, lymphocyte mitogenesis and NK activity in normal and cyclophosphamide immunosuppressed mice

The in vivo effect of isoprinosine on IL-2 production, mitogen-induced proliferation and NK activity of lymphocytes from normal as well as cyclophosphamide (CY) treated mice has been investigated. Isoprinosine was given in a single dose (50 mg/kg or 5 mg/kg) to normal or CY treated mice (250 mg/kg i.p. simultaneously to isoprinosine). An enhancement of T lymphocyte proliferation and IL-2 production was observed in both cases. There was no correlation between the small effect observed in T lymphocyte proliferation and the enhancement of IL-2 levels found in the supernatants of Con A activated spleen cells, specially in normal mice. Administration of isoprinosine every day (50 mg/kg) augmented Con A induced mitogenesis, IL-2 production and NK activity in animals treated with cyclophosphamide, but not in normal mice. Isoprinosine could be of interest in a combined treatment with immunosuppressants for the restoration of certain immune functions. The effect of isoprinosine on immune responses may be mediated in part by changes in IL-2 activity.     

Kinetic analysis of the immunopotentiating effect of the hypoxanthine analogue, NPT-15392, on the interleukin-2 production potential of human lymphocytes

Previous studies have shown that NPT-15392 (9-erythro-(2-hydroxy, 3-nonyl) hypoxanthine) enhances a variety of lymphoid functional activities including proliferative responses to various antigenic and mitogenic stimuli. In order to account at least partially for this immunopotentiation by NPT-15392, we examined the effects of this compound on interleukin-2 (IL-2) production by cultures of mitogen-activated human peripheral blood mononuclear cells (PBMC). Coculture of PBMC with NPT-15392 and concanavalin A (Con A) for 24 h resulted in significant increase of IL-2 in the supernatants of such cultures as compared with the IL-2 levels of control, non-NPT-treated, Con A-activated cultures. This enhancing effect was demonstrable with final culture concentrations of NPT-15392 ranging from 0.1 to 0.5 microgram/ml. Doses of NPT-15392 in excess of 5.0 micrograms/ml resulted in modest suppression of net IL-2 production. Pretreatment of PBMC with NPT-15392 for 2-4 h prior to activation with Con A was sufficient to achieve maximum enhancement of IL-2 production (20-40% average increase). Exposure of PBMC to NPT-15392 for longer periods (i.e. 24 h) did not result in higher levels of IL-2 production. NPT-15392 alone did not induce IL-2 synthesis at any of the doses employed and did not induce proliferation of the IL-2-dependent target cells used to quantitate IL-2 activity. Because of the multipotential role of IL-2 in the immune system, enhancement of IL-2 production by NPT-15392 may be a central pathway whereby this compound augments many lymphoid effector functions.     

Decreased mucosal interleukin-4 (IL-4) production in gut inflammation.

AIMS--To determine the prevalence of cells secreting interleukin-4 (IL-4) in the gut mucosa of children with chronic inflammatory bowel disease. METHODS--Mononuclear cells were isolated from intestinal biopsy specimens from control children (n = 10) and children with active inflammatory bowel disease (Crohn's disease n = 15, ulcerative colitis n = 9, indeterminate colitis n = 3). Spontaneous IL-4 production was then measured by SPOT-enzyme linked immunosorbant assay (ELISA) using a pair of non-competing anti-IL-4 monoclonal antibodies. The percentage of T cells in the isolated cells were also determined and the prevalence of IL-4 secreting cells calculated per 10,000 T cells. RESULTS--In control children the mean number of IL-4 secreting cells was 15.1 per 10,000 T cells. In Crohn's disease and ulcerative colitis the means were 5.3 and 5.2, respectively. In two children with indeterminate colitis numbers were also low. There was no difference in the percentage of T cells in the cell preparations isolated from each patient group. The reduction of IL-4 secreting cells in patients with Crohn's disease was not caused by steroids. CONCLUSIONS--In idiopathic inflammatory bowel disease IL-4 secreting cells are reduced in diseased mucosa.     

普乐可复对难治性肾病综合征患者血清IL-2、sIL-2R的影响及其临床意义

目的:研究普乐可复(FK506)对难治性肾病综合征患者血清IL-2、sIL-2R的影响及其临床意义。方法:应用酶联免疫吸附法检测难治性肾病综合征患者经普乐可复治疗前后血清IL-2、sIL-2R水平变化,并监测患者24小时尿蛋白、血浆白蛋白、血脂的变化。结果:难治性肾病综合征患者经普乐可复治疗前血清IL-2、sIL-2R水平均显著高于正常对照组(P<0.05)。治疗后血清IL-2、sIL-2R水平较治疗前明显下降(P<0.05)。治疗前24小时尿蛋白水平显著高于正常对照组(P<0.01),血浆白蛋白显著低于正常对照组(P<0.01),血脂水平显著高于正常对照组(P<0.01);与治疗前比较,治疗后24小时尿蛋白水平显著下降(P<0.05),血浆白蛋白水平显著升高(P<0.01),血脂水平显著下降(P<0.05)。治疗后组与正常组比较,除IL-2外,余各项指标均无显著性差异(P>0.05)。结论:在难治性肾病综合征患者体内存在IL-2、sIL-2R的异常,普乐可复对其有明确的抑制作用,从而调节T细胞活性,有效降低24小时尿蛋白,提高血浆白蛋白含量,降血脂,缓解难治性肾病综合征的病情。     

Cytokine regulation of human monocyte interleukin-1 (IL-1) production in vitro. Enhancement of IL-1 production by interferon (IFN) gamma, tumour necrosis factor-alpha, IL-2 and IL-1, and inhibition by IFN-alpha.

IL-1 production (secreted and cell-associated) was measured in monocyte cultures stimulated by a variety of agents in vitro. Monocytes either adherent to conventional plastic culture plates in serum-free conditions, or in suspension in culture medium containing serum were stimulated to produce IL-1 during culture. In non-adherent, serum-free conditions, monocytes produced very low or undetectable amounts of IL-1 during 20 h of culture. Lipopolysaccharide (LPS) induced equivalent amounts of secreted and cell-associated IL-1, although at very low concentrations more cell-associated IL-1 was produced. IL-1 production in response to LPS could be augmented by crude lymphokine, IFN-gamma, or tumour necrosis factor (TNF) alpha. TNF-alpha preferentially augmented the production of cell-associated IL-1 in LPS-stimulated cultures. TNF-alpha induced a significant amount of IL-1 (mainly cell-associated) directly but could also induce IL-1 secretion when combined with IL-2 or IFN-gamma, or when in the presence of serum. IL-2 acted synergistically with low concentrations of IFN-gamma or IL-1 to induce significant levels of IL-1 production. IFN-alpha did not induce any IL-1 production, but was a potent inhibitor of IL-1 production induced by a variety of stimuli. These results suggest that IL-1 production may be enhanced or reduced by different cytokines at concentrations likely to be found in chronic inflammatory lesions.     

Inactivation of human interleukin-2 (IL-2) by alpha 2-macroglobulin-trypsin complexes.

The loss of the biological activity of interleukin-2 (IL-2, T-cell growth factor) in the presence of alpha 2-macroglobulin-trypsin (alpha 2M.t) complexes has been investigated using an IL-2-dependent cloned 'cytotoxic' murine T-cell line. While reaction mixtures of native alpha 2M, aprotinin or methylamine-treated alpha 2M and IL-2 had no effect on IL-2 activity when incubated at 37 degrees for 5 h, alpha 2M.t (90 nM, [T]:[alpha 2M] = 0.8) inactivated IL-2 at a rate of one-sixth of that of the free enzyme. This effect was abolished by treatment of alpha 2M.t with aprotinin (MW 6500). Soybean trypsin inhibitor coupled to Sepharose 4B was capable of absorbing the IL-2 degrading activity from the trypsin solution. In contrast, alpha 2M.t treated with the solid-phase immobilized soybean trypsin inhibitor continued to inactivate IL-2, but did not degrade a macromolecular substrate (remazol-brilliant blue hide). Thus, IL-2 (MW 15,500) gains access to the active site of the alpha 2M-bound trypsin, resulting in a rapid loss of its biological activity. These observations offer an explanation for the in vitro immunosuppressive effects of alpha 2M.t.