Michael B. Prystowsky

Lymphokine regulation of inflammatory processes: Interleukin-4 stimulates fibroblast proliferation

While recent evidence from several laboratories has shown that interleukin-4 (IL-4) can act on a number of cells in addition to B lymphocytes, these have thus far been limited to cells of the hematopoietic lineage. Here we report that murine IL-4 promotes DNA synthesis in both primary and immortalized fibroblasts. Marked stimulation of [3H]thymidine incorporation was observed for primary skin fibroblasts or Balb/c3T3 cells stimulated with HPLC- or immunoaffinity-purified as well as recombinant IL-4. Responses to immunoaffinity and recombinant IL-4 were completely blocked with anti-IL-4 antibody. Similar dose/response relationships were observed for recombinant IL-4 on skin fibroblasts and an IL-4 responsive murine T cell tumor, suggesting that the receptors for this lymphokine on these cells is similar. Together, these results show that IL-4 can cause DNA synthesis by murine fibroblasts presumably through ligand-receptor interactions at the cell surface. Implications of these findings to inflammation during an immune response is discussed.

Recombinant interleukin 2 regulates levels of c-myc mRNA in a cloned murine T lymphocyte.

The cellular oncogene c-myc has been implicated in the regulation of growth of normal and neoplastic cells. Recently, it was suggested that c-myc gene expression may control the G0----G1-phase transition in normal lymphocytes that were stimulated to enter the cell cycle by the lectin concanavalin A (ConA). Here we describe the effects of purified recombinant interleukin 2 (rIL2) and of ConA on levels of c-myc mRNA in the noncytolytic murine T-cell clone L2. In contrast to resting (G0) primary cultures of lymphocytes, quiescent L2 cells have a higher RNA content than resting splenocytes and express receptors for interleukin 2 (IL2). Resting L2 cells are therefore best regarded as early G1-phase cells. Purified rIL2 was found to stimulate the rapid accumulation of c-myc mRNA in L2 cells. Levels of c-myc mRNA became maximal within 1 h and declined gradually thereafter. In contrast, ConA induced slower accumulation of c-myc mRNA in L2 cells, with increased levels of c-myc mRNA becoming detectable 4 to 8 h after stimulation. Experiments with the protein synthesis inhibitor cycloheximide demonstrated that the increase in levels of c-myc mRNA that were induced by ConA was a direct effect of this lectin and not secondary to IL2 production. Cyclosporin A, an immunosuppressive agent, markedly reduced the accumulation of c-myc mRNA that was induced by ConA but only slightly diminished the accumulation of c-myc mRNA that was induced by rIL2. Taken together, these data provide evidence that (i) c-myc gene expression can be regulated by at least two distinct pathways in T lymphocytes, only one of which is sensitive to cyclosporine A, and (ii) the accumulation of c-myc mRNA can be induced in T cells by IL2 during the G1 phase of the cell cycle.

Sexually dimorphic effects of alcohol exposure in utero on neuroendocrine and immune functions in chronic alcohol-exposed adult rats.

Maternal ethanol consumption has deleterious effects on the offsprings neuroendocrine and T-cell-dependent functions. Chronic alcohol consumption in adulthood has also been associated with activated hypothalamic-pituitary-adrenal (HPA) function and immunosuppression which is demonstrable at the T-cell level. Our aim was to establish whether prenatal alcohol exposure alters the neuroendocrine and immune responses to chronic alcohol challenge in adult male and female offspring. Adult male rats placed on a liquid alcohol diet for 5 weeks had significantly decreased thymus weight, hypertrophied adrenals, and elevated plasma ACTH and corticosterone levels. Splenic lymphocyte Concanavalin A (Con A)-stimulated proliferation in the ethanol-treated rats was decreased compared to that in pair-fed controls. Thus, prolonged alcohol exposure activated the HPA axis and suppressed T-cell function. The effects of prenatal alcohol exposure, as a predispositional factor, on the HPA axis and on the T-cell functions of adult chronic alcohol-exposed rats were examined in the offspring of dams fed ethanol (FAE) or an isocaloric liquid (PF) diet during the last 2 weeks of gestation. The adult offspring of both sexes and prenatal treatment groups were then placed on an alcohol-containing liquid diet for 5 weeks. Fetal alcohol exposure decreased anterior pituitary proopiomelanocortin mRNA levels and increased glucocorticoid receptor (GR) mRNA levels in males and decreased GR mRNA levels in females. There were no differences in hypothalamic CRF mRNA and GR mRNA levels between the prenatal treatment groups. There was no significant difference in Con A-stimulated lymphocyte proliferation between FAE and PF males. However, FAE females showed Con A-stimulated lymphocyte proliferation significantly higher than those of all other groups, including pair-fed females. Prostaglandin E(2) (PGE(2)) suppressed lymphocyte proliferation to a lesser degree in FAE males than in any other group. Furthermore, T-cell response to Con A was enhanced by indomethacin, a prostaglandin biosynthesis inhibitor, in FAE males suggesting that increased prostaglandin synthesis may occur in FAE males after chronic alcohol exposure. Increased levels of endogenous PGE(2) could also be inferred from the enhanced levels of interleukin-2 receptor alpha (IL-2Ralpha) mRNA in activated lymphocytes of male but not of female FAE offspring compared to PF. In summary, the results of this study demonstrate that prenatal alcohol exposure leads to a specific HPA-related vulnerability in males to the deleterious effects of ethanol in adulthood. Although prenatal alcohol did not further aggravate the effects of chronic alcohol exposure on lymphocyte proliferation response to Con A in adult male offspring, altered T-cell responses could be unmasked.

REGULATION OF GENE EXPRESSION IN LECTIN-STIMULATED OR LYMPHOKINE-STIMULATED T LYMPHOCYTES1

The effects of cyclosporine were examined on gene expression induced in T lymphocytes by mitogenic lectins and interleukin 2 (IL‐2). Used at concentrations that inhibited proliferation of human peripheral blood lymphocytes by ≈90%, CsA suppressed, to different extents, the phytohemagglutinin‐stimulated expression of various genes, with levels of mRNAs for IL‐2 being inhibited by ≈100%, c‐myc and N‐ras by ≈80%, and c‐fos and IL‐2 receptors by ≈50%. Comparisons of the actions of CsA on gene expression in a cloned murine T cell (L2), stimulated with concanavalin A or IL‐2, demonstrated that CsA specifically blocked the accumulation of mRNAs for the c‐myc and p53 protooncogenes when induced by Con A, but not when induced by IL‐2. Taken together, these findings indicate that several pathways can control the expression of a particular gene, and suggest that CsA interferes with only some of these regulatory pathways of gene expression in T lymphocytes.

Expression of prostaglandin GH synthase (cyclooxygenase) during murine fetal thymic development

Fetal thymic lobes in organ culture have been shown to have the capacity to metabolize [14C]arachidonic acid (AA) to prostaglandins (PGs), including 6-ketoPGF1 alpha, PGF2 alpha, PGE2, and PGA2. Inhibition of AA metabolism results in inhibition of growth and Thy 1 expression during thymic organ culture. We report herein that freshly-isolated fetal thymic lobes also have the capacity to metabolize [14C]AA to PGs and HETEs at Days 14 and 16 of prenatal murine development. RNA encoding phospholipase A2, which liberates arachidonic acid from membrane phospholipids, and cyclooxygenase (prostaglandin G/H synthase), the first enzyme involved in the conversion of AA to PGs, are expressed during thymic development. We have localized the cyclooxygenase protein to stromal cells in the fetal and adult thymus. Exogenous AA or an analogue of PGI2 (iloprost) stimulated growth of fetal thymocytes in organ culture. These findings, together with our studies of the morphology of thymic lobes cultured with inhibitors of arachidonate metabolism, support the hypothesis that PGs are required for thymocyte proliferation during thymic development.

Deletion of carboxy-terminal residues of murine granulocyte—Macrophage colony-stimulating factor results in a loss of biologic activity and altered glycosylation

A deletion mutant of murine granulocyte-macrophage colony-stimulating factor (GM-CSF) which differs in primary structure from native GM-CSF in the carboxy-terminal 11 amino acids was prepared. Four amino acid residues are mutated and the seven terminal residues including Cys-118 are deleted. Supernatants from COS-1 cells transfected with this deletion mutant (GM-CSF(del] showed a 3000-fold decrease in the ability to stimulate bone marrow stem cells to proliferate and differentiate into granulocytes and macrophages in vitro. Northern blot analysis using poly(A)+ RNA extracted from the transfected cells showed equal accumulations of GM-CSF and GM-CSF(del). Transfection with full-length GM-CSF followed by immunoprecipitation of metabolically labeled supernatant proteins with rabbit anti-rGM-CSF antiserum yielded predominantly the 23-kDa, fully glycosylated form and small amounts of both a 29-kDa form and the 18-kDa non-N-glycosylated form. Transfection of the GM-CSF(del) mutant and immunoprecipitation revealed a large, diffuse band on sodium dodecyl sulfate--polyacrylamide gel electrophoresis with a molecular weight of about 29 kDa. Digestion of the immunoprecipitated 29-kDa species with N-glycanase converted the 29-kDa form into two forms of about 23 and 18 kDa, suggesting that the increase in molecular weight of the deletion mutant protein resulted from hyperglycosylation. Adding tunicamycin to the culture medium of cells transfected with GM-CSF(del) also yielded a single non-N-glycosylated species of about 18 kDa, but secretion was at a significantly lower level than either the 29-kDa hyperglycosylated GM-CSF(del) protein from non-tunicamycin-treated cells or the 18-kDa non-N-glycosylated full-length GM-CSF from tunicamycin-treated cells. Since very recent scanning-deletion analysis indicates that there is a critical region for activity near Cys-118 and that Cys-118 is necessary for maximal activity, we conclude that the Cys-118 residue is necessary for proper glycosylation and maximal biologic activity of GM-CSF.

Environmental lighting alters the infection process in an animal model of AIDS.

In this study, we examined the effects of altered environmental lighting on the infection process of a murine leukemia virus, E-55(+), which induces a thymic lymphoma/leukemia in 100% of BALB.K mice inoculated as adults. One to two weeks after inoculation, high levels of proviral DNA are usually found. This is followed by an asymptomatic period of many weeks during which proviral DNA becomes essentially undetectable. Leukemia develops approximately 28 weeks postinoculation. In this experiment, one group of mice was exposed a consistent 10L: 14D cycle while a second was maintained in constant light (LL). A third group was exposed to a rotating cycle characterized by phase shifting a 10L: 14D cycle every three 24-h days (rLD). All cycles began 2 weeks prior to inoculation and were maintained thereafter. Animals were sacrificed at 1, 5, 10, and 15 weeks, and hematopoietic tissue was examined for proviral DNA content. At 1 week, LL- and rLD-exposed animals showed considerably less proviral DNA in bone marrow and spleen compared with controls. At 15 weeks, thymuses from controls were showing signs of infection whereas tissue from LL and rLD mice remained at background levels. We conclude that environmental lighting does alter the infective pattern displayed by this retrovirus, although whether this effect is mediated by changes in the target stem cells or through immunoenhancement has not yet been determined.

Evaluation of human N-linked glycosylation sites in murine granulocyte-macrophage colony-stimulating factor☆

Nonglycosylated murine and human granulocyte-macrophage colony-stimulating factor have a molecular mass of approximately 14.5 kDa predicted from the primary amino acid sequence. The expression of both proteins in COS cells leads to a heterogeneous population of molecules that differ in the degree of glycosylation. Both human and murine molecules contain two N-linked glycosylation sites that are situated in nonhomologous locations along the linear sequence. Despite this difference both proteins show a similar size distribution among the glycosylation variants. These studies analyze the effects of introducing in the murine protein novel N-linked glycosylation sites corresponding to those sites found in the human molecule. A panel of molecules composed of various combinations of human N-linked glycosylation sites in either the presence or the absence of murine N-linked glycosylation was compared. Substitution of a proper human N-linked glycosylation consensus sequence at Asn 24 did not result in N-linked glycosylation, nor was there any considerable effect on bioactivity. Replacement of the N-linked glycosylation consensus sequence at Asn 34 results in glycosylation similar to that found in the human molecule and causes a significant decrease in bioactivity. These data suggest that the position of N-linked glycosylation is critical for maximal bioactivity in a particular species and that the changes in position of these sites in different species probably occurred during evolution in response to changes in their receptors.

Evaluation of B-cell immunity in patients with pretransplant sensitization

The influence of presensitization (blood transfusions) on B-cell immunity as reflected in the serum of two groups of candidates for cadaveric donor renal allografts was examined. The first group initially had a high level of panel-reactive antibody (PRA) greater than 72% but experienced a large decrease in PRA (>70%) 6–34 months prior to transplantation. In contrast, the second group maintained a high PRA (100%) for up to 28 months after sensitization and before transplantation. Three blood samples from each patient, representing a maximum time span of 34 months, were analyzed. Levels of IgG, IgM isohemagglutinins, and antitetanus antibody were used as indicators of B-cell function. There were no significant differences between the individual values of a single patient with regard to each parameter. However, Group II patients had elevated values of total IgG relative to Group I patients. Total serum IgG-subclass levels (IgG1, IgG2, IgG3, IgG4) were measured and the relationship between a specific IgG subclass and the PRA activity was determined. IgG1 values in Group II were higher than those found for Group I. The other IgG subclasses were all within normal levels and were not significantly different between Group I and Group II. When IgG-subclass typing of PRA was performed, IgG1 accounted for most of the activity in both groups and a fall in PRA-specific IgG1 was associated with the reduced PRA observed in Group I. The data indicate that humoral immunity, as reflected by total and specific immunoglobulin levels, is intact in general in the two groups of presensitized renal allograft candidates examined and that any loss of PRA activity reflects a reduction in a specific immune response.

Regulation of Proto-Oncogene Expression During T Lymphocyte Activation and Proliferation

The proliferation of T lymphocytes is regulated in part through their membrane receptors for antigen and for T cell growth factor (interleukin 2, IL-2). Stimulation of resting T cells by specific antigen or by mitogenic cells (phytohemagglutinin, concanavalin A) in the presence of accessory cells bearing surface antigens encoded by genes in the major histocompatibility complex (MHC) locus induces T cells to express receptors for IL-2 and (some but not all T cells) to secrete this growth factor. The interaction of IL-2 with its cellular receptor on antigen- or lectin-activated T cells subsequently results in expression of transferrin receptors. Binding of serum transferrin to its receptor in late G1 phase of the cell cycle is then required for T cells to undergo the G1 → S phase transition, thereby initiating DNA synthesis and resulting ultimately in cell division. Other growth factors, including insulin and insulin-like growth factors, do not appear necessary, at least for short-term growth of T cells (reviewed in 1–3).