Differential effects of chlorpromazine on the in vitro generation and effector function of cytotoxic lymphocytes.

Allograft rejection represents a cytotoxic response mediated to a large degree by thymus-derived T lymphocytes (1). The study of such cell-mediated cytotoxic phenomena has been greatly facilitated by the discovery first noted by Hayry and Defendi (2) and Wunderlich and Cany (3), that a natural consequence of allogeneic stimulation in an unidirectional mixed lymphocyte culture (MLC) was the appearance of cytotoxic lymphocytes specific for antigens present on the stimulator cells. Subsequent studies have shown that such in vitro generation of cytotoxic lymphocytes was dependent on the proliferative response in an MLC (4), was genetically determined (5), and possibly required the interaction of several subpopulations of T cells (6). We now report that the surface active agent chlorpromazine: (a) inhibits allogeneic stimulation of the proliferative response in an MLC; (b) inhibits the MLC generation of cytotoxic lymphocytes, (c) has no effect on the recognition, binding, or lysis of target cells by already sensitized lymphocytes; and (d) blocks a postproliferative membrane-mediated event, independent of proliferation, and necessary for the MLC generation of cytotoxic lymphocytes.

Augmented uptake of neuraminidase-treated sheep red blood cells: participation of opsonic factors.

Neuraminidase from Vibrio cholerae (VCN) was used to treat sheep red blood cells (SRBC) which were then incubated in vitro with murine peritoneal macrophages. The uptake of VCN-treated SRBC by macrophages was greater than the uptake of SRBC not treated with VCN. SRBC opsonized with normal mouse serum (NMS) were taken up to a greater extent than untreated SRBC. SRBC treated with VCN and opsonized with NMS were phagocytosed to a greater extent than untreated SRBC, VCN-treated SRBC, or opsonized SRBC. Evidence demonstrated that factors in serum from normal C3H/HeJ mice augmented the uptake of VCN-treated SRBC in greater amounts than of normal SRBC. These findings were discussed in relation to the increased immunogenicity of neuraminidase-treated cells.

Cimetidine-induced augmentation of human lymphocyte blastogenesis by mitogen, bacterial antigen, and alloantigen.

The effect of Cimetidine, a histamine-type 2 receptor antagonist, was evaluated on the in vitro proliferative response of normal human peripheral blood lymphocytes (PBLs). Cimetidine (10(-3) to 10(-8) M) increased mitogen-induced blastogenesis by 22% (phytohemagglutinin (PHA) and by 27% (pokeweed) over nondrug-treated control values (P less than 005 for PHA and pokeweed). Preincubation of PBLs with Cimetidine further augmented blastogenesis as much as 2- to 3-fold (P less than 0.005 for both mitogens). Multiple testing of the same normal subject demonstrated consistent reproducibility of increased proliferation by Cimetidine. Similar statistically significant amplifications of the proliferative res-ponse were observed when bacterial antigen (streptokinase-streptodornase) or alloantigen was used to induce blastogenesis. Optimally effective concentrations of Cimetidine ranged from 10(-5) to 10(-7) M, which corresponds to expected clinical serum levels. These observations suggest that a histamine-type 2 receptor antagonist is capable of modulating the proliferative response of PBLs in the absence of exogenously added histamine. The immunoregulatory implication of this Cimetidine-induced proliferative augmentation is discussed in relation to clinical transplantation and cancer immunotherapy.

Differential susceptibility of human peripheral blood lymphocyte subpopulations to mitogenic activation. Influence of methylprednisolone.

The inhibition of the mitogenic activation of human peripheral blood lymphocyte (PBL) subpopulations by methylprednisolone (MP) was dependent on the mitogen used. Purified human T cells were more sensitive to the effects of MP than were B cells or PBLs, especially when these cells were activated by pokeweed mitogen (PWM). MP did not function by inhibiting binding of mitogen to the cell surface. After being mitogenically activated, human lymphocytes were resistant to the effects of MP. These effects of MP were shown to be reversible. Monocytes did not provide a significant degree of protection to mitogenically activated human T cells incubated with MP. These data suggest that MP-induced inhibition of the mitogenic activation of human PBLs may be a reflection of lymphocyte heterogeneity, and that the differential sensitivity of PBLs to MP may be used to isolate functionally different subpopulations of these cells.

Histamine type-2 receptor antagonist immune modulation. I. Increased cell-mediated cytotoxicity in normal and in down-regulated systems.

Cimetidine, a histamine type-2 receptor antagonist, is capable of immunoregulating T cell-mediated proliferative responses in both man and mouse. We present data that show that cell-mediated cytotoxicity (CMC) is also increased by cimetidine in normal and down-regulated murine splenocytes. Timed addition and removal of cimetidine from CMC generation cultures localizes the cimetidine effect to the first 24 hours of the alloantigen sensitization process. Cimetidine partially reverses CMC suppression by suppressor cells generated in vitro in a dose response manner; the result depends on the number of suppressor cells added. Mixed tumor lymphocyte cytotoxicity of splenocytes from syngeneic-tumor-bearing mice is increased by in vivo cimetidine treatment, which results in prolongation of concomitant tumor immunity. These data support the concept that cimetidine may have potential as a clinical immunofacilitator in down-regulated states of immunity.

Improved renal allograft survival with selected HLA antigen matching.

We examined the influence of the three most frequent linkage disequilibrium antigen combinations, HLA 1-8, 2-12, and 3-7, on renal allograft survival. We reviewed the results of 214 first transplants to recipients sharing exactly two HLA-A or -B antigens with the donor, excluding failures caused by hyperacute rejection or technical factors. Actuarial graft survival for matches having HLA 1-8, 2-12 or 3-7 was compared with all other two-antigen matches within the group. Improved graft survival, occurred with HLA 1-8 matches (85% and 80% at 2 and 5 years, respectively) and with HLA 2-12 matches (80% at both 2 and 5 years), compared to all other two-antigen matches (71% and 58% at 2 and 5 years, respectively). Allografts matched for HLA 3-7 had poorer graft survival (44% at both 2 and 5 years). We compared nondiabetic and diabetic recipients and found similar improved graft survival with HLA 1-8 or 2-12 matches in nondiabetic recipients. There was no difference, however, in graft survival in diabetic recipients with HLA 1-8 or 2-12 matches, as compared to other two-antigen matches. We conclude that HLA antigen matches of 1-8 or 2-12 between donor and recipient have demonstrated improved graft survival in nondiabetic recipients, while HLA 3-7 matches have poorer survival, as compared to other two-antigen matches. Thus, unlike single antigen serotyping, selected HLA antigen matching appears to afford superior graft survival.

Effects of methylprednisolone on concanavalin A-induced human lymphocyte blastogenesis: a comparative analysis by flow cytometry, volume determination and 3H-thymidine incorporation.

The inhibition of concanavalin A-induced human peripheral blood lymphocyte blastogenesis by methylprednisolone (MP) was studied by using flow cytometry and tritiated thymidine (3H-TdR) incorporation. Flow cytometric determinations of volume, low angle forward light scatter, and nucleic acid showed MP to be a potent inhibitor of blastogenesis. The effects were concentration-dependent and correlated with 3H-TdR uptake. By using the single cell analytic capability of flow cytometry, the target stages of the cell cycle where MP affects lymphocyte activation were determined. Evidence is presented that steroids can block both early and late phases of this process.

Activation of purified human thymus-derived (T) cells by mitogens. II. Monocyte- macrophage potentiation of mitogen-induced DNA synthesis.

Thymus-derived (T) cells from peripheral blood were purified by rosette formation with neuraminidase-treated sheep red blood cells (SRBC) and centifugation on Ficoll-Hypaque. T cells recovered from the pellet were freed of SRBC by treatment with Tris-NH4Cl. T cells purified by this method showed a diminished ability to take up 3H-thymidine (3H-TdR) after mitogen stimulation when compared to the mitogenic response of an equal number of autologous peripheral blood mononuclear lymphocytes (PBL). Autologous monocytes restored the capacity of purified T cells to take up 3H-TdR in the presence of phytohemagglutinin (PHA) or Concanavalin A (Con A). The effect was proportional to the number of monocytes added. Similar restorative effects could be obtained with allogeneic or xenogeneic monocytes. These data suggest that the mitogenic stimulation of human PBL and Con A may reflect the participation of more than one cell type: the T cells and monocyte and that the genetic origin of the monocyte is not critical for augmentation of the mitogenic activation of human T cells.

Activation of purified human T cells by mitogens: diminished mitogen-induced deoxyribonucleic acid synthesis in human T cells compared with autologous peripheral blood lymphocytes.

Human thymus-derived (T) cells were isolated from peripheral blood after rosette formation with neuraminidase-treated sheep erythrocytes (SRBC). After separation on Ficoll-hypaque, SRBC were removed from T cells by treatment with tris(hydroxymethyl)aminomethane-NH4Cl. Human T cells and autologous peripheral blood lymphocytes (PBL) were then incubated with phytohemagglutinin, concanavalin A, or pokeweed mitogen. Human T cells, in the absence of other cell types, responded with less deoxyribonucleic acid (DNA) synthesis (measured by uptake of [3H]thymidine) than equal numbers of autologous PBL. Further experimentation established that, compared with autologous PBL, the diminished capacity of human T cells to be activated by mitogens was due neither to differences in the mitogen dose-response relationship nor to the time of peak DNA synthesis of T cells or autologous PBL. Fragments or components of SRBC were not detected on human T cells, and treatment of the T cell-SRBC mixture with tris(hydroxymethyl)aminomethane-NH4Cl did not contribute to the results. Increased cell density or a period of preculture before addition of mitogen also did not influence the degree of decreased DNA synthesis in human T cells compared with the response of autologous PBL incubated with the same mitogens. When mixed with the cells remaining at the Ficoll-media interface, purified human T cells did not suppress the mitogenic response of this cell mixture to PHA or concanavalin A. The data indicated that human T cells, in the absence of other cell types, were activated by mitogens to a lesser degree than autologous PBL. Furthermore, T cells responded with DNA synthesis after direct T cell-mitogen interaction.

Synergistic effects of lipopolysaccharide on phytohemagglutinin- and concanavalin A-induced deoxyribonucleic acid synthesis in human peripheral blood lymphocytes: participation of T lymphocytes.

Lipopolysaccharide induces a synergistic uptake of tritiated thymidine in peripheral blood lymphocytes (PBL) when cultured with phytohemagglutinin or concanavalin A as compared to PBL incubated only with phytohemagglutinin or concanavalin A. In this study we investigated which subpopulations(s) of PBL is involved in this synergistic increase in deoxyribonucleic acid synthesis. Separation of PBL into sheep erythrocyte rosette-forming cells (T cells) and non-rosette-forming cells (B cells) showed that the T cells were responsible for the increased uptake of radiolabel. PBL and T cells had similar dose-response profiles and kinetic patterns. Paralleling this augmented tritiated thymidine uptake was an increase in the number of cells undergoing blast transformation. Delayed-addition experiments showed that the two mitogens must be added within 12 h of each other for maximal augmentation to occur. Finally, preincubation of T cells with lipopolysaccharide had no demonstrable effect on the amount of concanavalin A uptake by these cells. This model may provide unique information about the activation of human peripheral blood T cells compared to activation of these cells by one mitogen.