Leucyl-leucine methyl ester treatment of donor cells permits establishment of immunocompetent parent----F1 chimeras that are selectively tolerant of host alloantigens

Treatment of murine lymphocytes with L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) selectively removes natural killer cells, cytotoxic T lymphocyte precursors, and the capacity to cause lethal graft-vs-host disease, whereas bone marrow stem cell function and alloantigen-induced L3T4+ T helper function remains intact. The present studies assess the immunocompetence of allogeneic bone marrow chimeras established by reconstituting irradiated (C57BL/6 X DBA/2)F1 (B6D2F1) mice with Leu-Leu-OMe-treated C57BL/6 (B6) bone marrow and spleen cells. Spleen cells from such chimeras were found to have normal B and T cell mitogenic responses. Furthermore, levels of natural-killer cell function were comparable to those observed in B6----B6 syngeneic radiation chimeras established without Leu-Leu-OMe treatment of donor cells. Spleen cells from B6----B6D2F1 mice were identical with B6----B6 or B6 mice in allostimulatory capacity and thus contained no discernible cells of non-H-2b phenotype. Whereas B6----B6D2F1 spleen cells demonstrated alloproliferative and allocytotoxic responses toward H-2k bearing spleen cells, no H-2d specific proliferative or cytotoxic responses could be elicited. B6----B6D2F1 spleen cells did not suppress the generation of anti-H-2d or anti-H-2k proliferative or cytotoxic responses from control B6 spleen cells. Furthermore, addition of rat concanavalin A supernatants did not reconstitute anti-H-2d responses of B6----B6D2F1 chimeric spleen cells. Thus, Leu-Leu-OMe treatment of B6 donor cells not only prevents lethal graft-vs-host disease, but also permits establishment of long-lived parent----F1 chimeras that are selectively tolerant of host H-2 disparate alloantigens, but fully immunocompetent with respect to natural killer cell function, B and T cell mitogenesis, and anti-third party alloresponsiveness.     

T cell-dependent activation of B cell proliferation and differentiation by immobilized monoclonal antibodies to CD3

The capacity of mAb directed at the CD3 molecular complex (64.1) to induce T cell-dependent B cell proliferation and differentiation was examined. Coculture of B cells with mitomycin C-treated T4 cells (T4 mito) stimulated by immobilized 64.1 resulted in marked B cell proliferation and Ig-secreting cells (ISC) generation in the absence of any additional stimulation. The magnitude of the B cell responses induced by immobilized 64.1-stimulated T4 mito was far greater than that induced by other stimuli, such as Staphylococcus aureus plus factors produced by mitogen-activated T cells, PWM, or soluble 64.1. The induction of maximal B cell responsiveness required direct contact between activated T cells and responding B cells. Of note, immobilized 64.1 also induced B cell proliferation and ISC generation in the presence of mitomycin C-treated T8 cells. By contrast, immobilized 64.1 stimulated T4 or T8 cells that had not been treated with mitomycin C induced very modest ISC generation and suppressed B cell responses supported by T4 mito even in the presence of exogenous IL-2 or factors produced by mitogen-activated T cells. The interactions between T and B cells in these cultures not only induced B cell responses, but also enhanced the production of IL-2 by activated T cells. Increased IL-2 production was facilitated when culture conditions afforded the opportunity for contact between B cells and activated T cells. These results indicate that the establishment of interactions between B cells and anti-CD3-stimulated T4 or T8 cells provides all of the signals necessary for proliferation and differentiation of B cells without other stimuli and also augments the production of lymphokines by the activated T cells. The data emphasize the role of Ag-nonspecific interactions between B cells and T cells in promoting polyclonal responses of both cell types.     

Leu-Leu-OMe sensitivity of human activated killer cells: delineation of a distinct class of cytotoxic T lymphocytes capable of lysing tumor targets

Sensitivity to L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) was used to characterize the phenotype of human activated killer cells. Natural killer cells (NK) and the precursors of both the alloantigen-specific cytotoxic T lymphocytes (CTL) and the NK-like activated killer cells generated after stimulation with allogeneic cells were deleted from human peripheral blood lymphocytes by preincubation with Leu-Leu-OMe. It was noted, however, that cytotoxic lymphocytes could be generated from Leu-Leu-OMe-treated lymphocyte precursors after 2 to 6 days of culture with the nonspecific mitogen, phytohemagglutinin (PHA). The characteristics of these killer cells indicated that they were a unique population that could be distinguished from other cytotoxic cells. Killing by these cells exhibited slow kinetics in that 18 hr cytotoxicity assays were required to detect full cytotoxic potential. When 18 hr assays were used, PHA-stimulated cytotoxic cells generated from Leu-Leu-OMe-treated lymphocytes were able to kill both NK-sensitive K562 cells and the relatively NK-resistant renal cell carcinoma cell line, Cur. These cytotoxic lymphocytes were HNK-1, Leu-11b (CD16), and OKM1 (CR3)-negative at both the precursor and effector stage of activation. Furthermore, these cells were derived from a CD3-positive precursor. Finally, killing by activated effectors was inhibited by OKT3. Unlike activation of Leu-Leu-OMe-sensitive large granular lymphocytes, generation of these cytotoxic T cells was totally prevented by treatment with mitomycin c before stimulation. Thus, a unique class of tumoricidal T cells can be characterized by resistance of lymphocyte precursors to a concentration of Leu-Leu-OMe, which has been shown to ablate NK, mixed lymphocyte culture-activated NK-like cytotoxic precursors, and the precursors of alloantigen-specific CTL.     

Comparison of trout hepatocyte culture on different substrates

Summary Comparisons were made of attachment and viability of rainbow trout (Salmo gairdneri) hepatocytes in short-term (2 days), primary culture on plastic, collagen-coated or extracellular matrix (ECM) coated dishes. Hepatocyte isolation routinely yielded cells with good viability (96%). Cells plated on ECM attached with high efficiency (93%) in contrast to cells cultured on plastic or collagen (∼20%). The cells plated on ECM flattened out and formed monolayers, while the cells on plastic and collagen rounded up and formed multi-cell aggregates in suspension. Viability of cells in all substrates remained high over the 2 day culture period. ECM is the first substrate to support trout-hepatocyte attachment in primary culture. Differentiated liver function was maintained in cells cultured on ECM as evidence by the induction of tyrosine aminotransferase by hydrocortisone (200%). This work was supported in part by research grant R809599010 from the U. S. Environmental Protection Agency. Editor's Statement This paper reports improved methods for culture of trout liver-derived cells that make in vitro investigations of fish metabolism, carcinogenesis and chemical toxicity more feasible than previously applied techniques. Recent interest in fish as models for study and indicators of effects of envionmental and food-related toxins make this work timely, poarticularly since many of the compounds of interest are primarily metabolized by hepatocytes or act on liver as a major target. David W. Barnes     

The four biochemically distinct species of human interleukin 1 all exhibit similar biologic activities

The supernatants of human monocytes incubated with endotoxin are able to stimulate the proliferation of murine thymocytes in the presence of PHA. This is known as LAF (lymphocyte activating factor) activity and is a characteristic activity of interleukin 1 (IL 1). The LAF activity can be resolved into four major fractions: a 15,000 dalton (pI 7), a 15,000 (pI 5.5), a 35,000 (pI 7), and a 35,000 (pI 5.5) fraction. To determine whether these four fractions shared the other biologic activities ascribed to IL 1, they were compared in a series of bioassays. When standardized with respect to their LAF activities, the four fractions did not differ significantly as mitogens for murine thymocytes, inducers of IL 2, murine or human B cell activators, human chondrocyte or synoviocyte stimulants, or inducers of acute phase proteins in vivo. On the other hand, the samples differed markedly as stimulators of porcine synoviocytes, with the 15,000 dalton (pI 5.5) fraction being the only strongly active fraction. These results are consistent with the hypothesis that all four LAF could be products of a single gene, although the porcine receptor may be able to distinguish between them. If this is the case, all four fractions can properly be termed IL 1.     

Immunologic function of endothelial cells: guinea pig aortic endothelial cells support mitogen-induced T lymphocyte activation, but do not function as antigen-presenting cells

The possibility that vascular endothelial cells (EC), like macrophages (M phi), can function as accessory cells necessary for mitogen- and antigen-induced T cell activation was examined. EC were enzymatically detached from the luminal surfaces of guinea pig aortas and then propagated in culture. Lymph node T lymphocytes were rigorously depleted of adherent cells, such that they completely lost the capacity to respond to mitogenic stimulation with phytohemagglutinin or concanavalin A. In this system, EC restored mitogen-induced T cell DNA synthesis as effectively as did M phi. This effect could not be explained by a facilitation of residual accessory cell activity within the responding T cell population, because EC restored mitogen responsiveness to T cells that had been treated with anti-Ia antibody and complement. Support of mitogen responsiveness could not be accounted for by secreted products of M phi or EC in the absence of intact accessory cells. In addition to the capacity to serve as fully sufficient accessory cells for the induction of mitogen-stimulated T cell proliferation, EC exerted a number of modulatory influences on T lymphocyte responses in cultures supported by M phi. When such cultures were supplemented with small numbers of EC, responses were dramatically augmented; larger numbers of EC resulted in marked suppression. At least part of these immunomodulatory effects could be accounted for by the activity of secreted products of EC. EC did not express detectable Ia antigens assayed either by indirect immunofluorescence, with the use of the fluorescence-activated cell sorter, or by complement-mediated cytotoxicity. Moreover, treating the EC population with anti-Ia antibody and complement had no effect on its capacity to support mitogen-induced T cell DNA synthesis. As would be expected from the lack of Ia antigen expression, EC were incapable of presenting antigen to primed T cells. They did, however, carry enough antigen into the cultures such that effective antigen presentation could occur when the cultures were supplemented with M phi that were syngeneic but not allogeneic to the responding T cells. Moreover, EC were capable of dramatically augmenting antigen-specific responses stimulated by antigen-pulsed M phi. There was no genetic restriction for this EC-mediated augmentation of antigen responsiveness. These results indicate that EC are capable of functioning as completely sufficient accessory cells for mitogen-induced T cell DNA synthesis and, in addition, are able to modulate ongoing M phi-supported T lymphocyte responses in both a positive and negative manner.(ABSTRACT TRUNCATED AT 400 WORDS)     

The role of interleukin 1 in human B cell activation: inhibition of B cell proliferation and the generation of immunoglobulin-secreting cells by an antibody against human leukocytic pyrogen

The role of factors released by monocytes (M phi) in the activation of human B lymphocytes was examined by studying the effect of an antiserum against human leukocytic pyrogen (LP) on mitogen-stimulated B cell proliferation and the generation of immunoglobulin-secreting cells (ISC) by peripheral blood mononuclear cells (PBM). Antiserum against LP was obtained from rabbits immunized with LP-containing human M phi supernatants. The globulin fraction of this antiserum inhibited pokeweed mitogen- (PWM) stimulated B cell proliferation and the generation of ISC in a concentration-dependent manner, with 50% inhibition of responsiveness observed with 10 micrograms/ml. By contrast, PWM-induced T cell [3H]thymidine incorporation was not inhibited by concentrations of anti-LP as great as 2000 micrograms/ml. The F(ab')2 fraction of anti-LP also inhibited the generation of ISC in response to both PWM and formalinized Staphylococcus aureus, but required 50 micrograms/ml to achieve 50% inhibition. Anti-LP inhibited the generation of ISC only if present during the first 24 hr of a 6 to 7-day incubation; later addition was not inhibitory. Inhibition was more marked in cultures partially depleted of M phi than in whole PBM cultures. Whereas absorption of the anti-LP with PBM failed to remove the capacity to inhibit the generation of ISC, anti-LP-mediated inhibition of responsiveness could be reversed by the addition of crude M phi culture supernatants or a variety of highly purified interleukin 1 (IL 1) preparations, but not by T cell supernatants. These results indicate anti-LP inhibits human B cell activation by removing the requisite M phi-derived factor IL 1 and also confirm that IL 1 plays an essential role in B cell proliferation and the generation of ISC in man.     

70-Kilodalton Heat Shock Protein Induction in Cerebellar Astrocytes and Cerebellar Granule Cells In Vitro: Comparison with Immunocytochemical Localization After Hyperthermia In Vivo

Induction of the 70-kDa heat shock protein, hsp70, was evaluated in cultured cerebellar astrocytes and granule cell neurons subjected to a hyperthermic stress, using a monoclonal antibody and an oligonucleotide probe that selectively recognize stress-inducible species of hsp70-related proteins and RNAs, respectively. Immunoblots of cultures enriched in either granule cells or astrocytes, and immunocytochemical localization studies in cocultures of these cell types, demonstrated that hsp70 induction was restricted to the astrocyte population. Amino acid incorporation experiments showed little difference in the loss and recovery of overall protein synthesis activity in these two cell types following transient hyperthermic stress. RNA blot hybridizations confirmed the preferential glial induction of hsp70. In vivo immunocytochemical studies in brains of adult rats following hyperthermia were consistent with earlier observations that suggested a primarily glial and vascular localization of the heat shock response in most brain regions, although the intense immunoreactivity in the cerebellar granule cell layer suggests that there is induction of hsp70 in these neurons under in vivo conditions. These results suggest the potential value of such defined cell cultures in identifying mechanisms responsible for differences in the heat shock response of various cell types in vitro, and in revealing factors that may account for the apparent absence of the stress response in cultured cerebellar granule cell neurons.     

Lipoproteins may provide fatty acids necessary for human lymphocyte proliferation by both low density lipoprotein receptor-dependent and -independent mechanisms

Human lymphocytes respond optimally to mitogenic stimulation when cultured in serum-free medium supplemented with transferrin if fatty acids necessary for maximal proliferation are provided. Either lipoproteins or exogenous fatty acids support optimal lymphocyte responses. The current studies examined the role of cell surface receptors for low density lipoprotein (LDL) in the enhancement of lymphocyte proliferation. Support of lymphocyte growth by limiting concentrations of LDL was found to involve interaction of the lipoprotein with LDL receptors. Thus, modification of LDL by reductive methylation so as to inhibit receptor-mediated interactions markedly decreased the capacity of LDL to enhance lymphocyte proliferation. Moreover, growth of lymphocytes obtained from patients with LDL receptor-negative homozygous familial hypercholesterolemia was minimal when cultures were supplemented with low concentrations of LDL (less than 10 micrograms cholesterol/ml). LDL also enhanced lymphocyte proliferation by a receptor-independent mechanism since high concentrations (greater than or equal to 50 micrograms cholesterol/ml) supported growth of both normal and familial hypercholesterolemia lymphocytes. In contrast, support of lymphocyte proliferation by high density lipoprotein (HDL) subclass 3 was completely independent of LDL receptors. Thus, HDL3 enhanced responses of both normal and familial hypercholesterolemia lymphocytes in an equivalent concentration-dependent manner; this effect was not altered by reductive methylation of HDL3. One function of lipoproteins in this system may be the provision of fatty acids since oleic and linoleic acids enhanced DNA synthesis by both normal and familial hypercholesterolemia lymphocytes in the absence of lipoproteins. These results indicate that lipoproteins may provide fatty acids necessary for optimal proliferation of human lymphocytes by both LDL receptor-mediated and LDL receptor-independent interactions.