Showing papers in "European Journal of Immunology in 1988"

Establishment of mouse cell lines which constitutively secrete large quantities of interleukin 2, 3, 4 or 5, using modified cDNA expression vectors

Abstract: Mouse cell lines of different lineages have been established which constitutively secrete large quantities of recombinant mouse interleukins (mIL2, mIL3, mIL4 or mIL5). An existing bovine papilloma virus-based expression vector, pBV-1MTHA, was modified to allow transformed X63Ag8-653 myeloma cells, NIH 3T3 fibroblasts and C127 mammary tumor cells to stably carry multiple copies of the vector, to express the inserted cDNA encoding a single interleukin constitutively, and to secrete the interleukin in high quantities. Cell lines transformed with mIL2 cDNA stably carried 30-100 copies of the plasmid per cell and constitutively secreted biologically active mIL2 in quantities similar to those produced by murine EL4 thymoma cells or rat spleen cells stimulated with mitogens. Deletion of the 3' untranslated region containing AT-rich sequences from the mIL2 cDNA resulted in a 100-fold increase in the constitutive production and secretion of mIL2 by the transformants. Addition of a heavy metal further increased the production 2 to 6-fold. Cells transformed with 3'-deleted mIL3 cDNA constitutively secreted 300-1000 times higher activities of mIL3 than the myelomonocytic leukemia line WEHI3. mIL4 produced by the similar transformants induced [3H]thymidine uptake of a T cell line, a mast cell line and B leukemia cells, and enhanced the production of IgG1 by B cells. IL4 titers were 150 times higher than those produced by the concanavalin A-stimulated T cell line 2.19. mIL5 was secreted by similar transformants at 10-fold higher titers than those produced by concanavalin A-stimulated 2.19 T cells, as judged by the proliferation and maturation of B cell leukemia BCL1. The expression vectors should be useful in establishing eukaryotic cell lines producing proteins from full length cDNA clones at higher rates. The established cell lines secreting IL2, 3, 4 or 5 at high rate should be useful sources for these interleukins in the investigation of their function in the immune system.

Excessive production of interleukin 6/B cell stimulatory factor‐2 in rheumatoid arthritis

Abstract: High levels of interleukin 6 (IL 6/B cell stimulatory factor-2) were detected in synovial fluids from the joints of patients with active rheumatoid arthritis (RA). The cells found in freshly isolated synovial fluid constitutively expressed IL 6 mRNA. The synovial tissues obtained by joint biopsy were also found to produce IL 6 in vitro. Immunohistochemical analysis demonstrated that CD2+ T cells as well as CD20+ blastoid B cells in the synovial tissues produce IL 6. The data indicate that IL 6 is generated constitutively in RA and its overproduction may explain the local as well as the generalized symptoms of RA, since IL 6 can function as B cell growth and differentiation factor as well as hepatocyte-stimulating factor.

Interferon-γ and tumor necrosis factor induce the L-arginine-dependent cytotoxic effector mechanism in murine macrophages*

Abstract: We tested several monokines and muramyl dipeptide (MDP) to determine whether they induce the L-arginine-dependent effector mechanism in cultured murine macrophages. Recombinant interferon-gamma (rIFN-gamma) and recombinant tumor necrosis factor (rTNF) synergize to induce nitrite (NO2-) and nitrate (NO3-) synthesis from L-arginine as well as to cause inhibition of the iron-dependent enzyme aconitase in macrophages. Unlike rTNF, recombinant interleukin 1 (rIL 1) and rIL 6/B cell stimulatory factor 2 (rIL 6/BSF-2) did not act as cofactors when added to macrophages in the presence of rIFN-gamma. rIFN-gamma plus MDP induced the L-arginine-dependent effector mechanism in murine macrophages. However, induction by rIFN-gamma plus MDP was inhibited by anti-rTNF antibodies which suppressed both NO2-/NO3- synthesis and aconitase inhibition. This result indicates that endogenously produced TNF is involved in the induction of the L-arginine-dependent effector mechanism when MDP is the co-stimulant with rIFN-gamma. In contrast, anti-rTNF antibodies did not fully suppress the effect of combining rIFN-gamma and lipopolysaccharide, suggesting that, in this case, activation of the L-arginine-dependent effector pathway may involve more than induction of TNF synthesis by the macrophages. These results provide information, at a biochemical level, on a mechanism through which combination of IFN-gamma and TNF can modulate macrophage functions involved in the control of cell proliferation.

The half-lives of serum immunoglobulins in adult mice

Abstract: We determined the half-lives of several sets of murine monoclonal antibodies spanning all immunoglobulin isotypes in the serum. The antibodies in each set possess the same V region. With this approach, the differences in half-life observed between the different isotypes are independent of the V region carried by the monoclonal antibodies and therefore must relate to each other in the same way as the half-lives of each class of serum immunoglobulins. The half-life of a monoclonal antibody of the gamma 2a isotype is identical to the average half-life of serum IgG2a as previously determined (6-8 days; P. Vieira and K. Rajewsky, Eur. J. Immunol. 1986. 16:871). Therefore, the half-lives determined with monoclonal antibodies possessing the same V region represent the half-life of the serum immunoglobulins. In this way we calculated the half-life of IgM as 2 days, IgG3 and IgG1 as 6-8 days, IgG2b has a half-life of 4-6 days. IgE has a half-life of 12 h. A polymeric form of IgA was found to be eliminated from the serum with a half-life of 17-22 h.

Functional discrimination between interleukin 6 and interleukin 1

Abstract: In this study we have investigated the specificity of bioassays in which interleukin (IL)1 and/or IL6 are active. The thymocyte assay cannot be used to discriminate between IL1 and IL6; both monokines are active in this assay. Moreover the detection limit for both IL1 and IL6 is around 100 pg/ml. IL6 activity can be measured with a murine hybridoma cell line (B9). The detection limit for human as well as murine IL6 is about 0.5 pg/ml. The assay is specific for IL 6 and is not influenced by a variety of other cytokines except for murine IL4 which shows some activity in this assay. IL1 can be measured specifically with D10 cells. The detection limit for IL1α and IL1β is around 1 pg/ml whereas IL6 is not active in this assay at all. Upon stimulation by IL1 and/or IL2 D10 cells produce IL6. However, this IL6 does not seem to be involved in the proliferation of these cells.

Interleukin 6 is involved in interleukin 1-induced activities

Abstract: Human monocytes produce a number of soluble mediators involved in regulation of inflammation and lymphocyte growth and differentiation such as interleukin 1 (IL 1) and tumor necrosis factor. Recently, the cDNA of another monocyte-derived factor, interleukin 6 (IL 6), was cloned. Herein we show that purified E. coli-derived recombinant IL 6 (rIL 6) is as active as IL 1 in the thymocyte assay. In addition, IL 1 and IL 6 synergize strongly in stimulating thymocyte proliferation. Another property shared by IL 1 and IL 6 is their pyrogenicity. Human rIL 6 induces a monophasic fever after i.v. injection into rabbits. Together with the observation that IL 1 induces IL 6 in a variety of cells including thymocytes, our data suggest that IL 6 is involved in many of the pleiotropic effects of IL 1.

Induction of rat acute-phase proteins by interleukin 6 in vivo.

Abstract: Recombinant human interleukin 6 (rhIL 6) was injected i.p. into male Wistar rats to investigate its role as a mediator of the acute-phase response. Hepatic mRNA levels of beta-fibrinogen, alpha 2-macroglobulin, cysteine proteinase inhibitor, alpha 1-acid glycoprotein and albumin were measured at different times after the administration of rhIL 6. Maximal increases of mRNA concentrations were observed already 4 h after the injection of rhIL 6 leading to 4.8-, 19.7-, 10- and 16-fold stimulations in mRNA levels of beta-fibrinogen, alpha 2-macroglobulin, cysteine proteinase inhibitor or alpha 1-acid glycoprotein, respectively. The rhIL 6-induced stimulation of acute-phase protein mRNA was much more rapid than the acute-phase induction after turpentine, where maximal mRNA levels were found between 16 and 24 h. For all acute-phase proteins studied, the stimulation of mRNA synthesis was found to be dependent on the dose of rhIL 6 injected. In the case of alpha 2-macroglobulin mRNA a sex-specific induction by rhIL 6 was found. Only male rats showed an acute-phase response, whereas in female rats an acute-phase reaction of alpha 2-macroglobulin mRNA was not inducible by IL 6. The increases in mRNA levels of the acute-phase proteins studied were followed by corresponding changes of the proteins in the serum determined by rocket immunoelectrophoresis. It is concluded that IL 6 represents a potent mediator of the acute-phase response in the rat.

Human Ig superfamily CTLA-4 gene: chromosomal localization and identity of protein sequence between murine and human CTLA-4 cytoplasmic domains.

Abstract: The mouse CTLA-4 gene has been shown to code for an activated lymphocyte-associated sequence belonging to the Ig gene superfamily. We now report on the molecular cloning and study of the human corresponding gene isolated from a genomic library and designated Hu-CTLA-4. The Hu-CTLA-4 gene exists as a single copy per human haploid genome and maps to band q33 of chromosome 2. It comprises 3 exons notwithstanding the leader sequence. The first exon encodes a V-like domain of 116 amino acids, the second one a hydrophobic putative transmembrane region of 37 amino acids and the third one a 34 amino acid putative cytoplasmic domain. Whereas the overall homology between the human and murine CTLA-4 proteins is 76%, there is, remarkably, a complete identity of their cytoplasmic domains. This complete interspecies conservation comes in support of an important role for this domain in CTLA-4 function.

The function of human intercellular adhesion molecule‐1 (ICAM‐1) in the generation of an immune response

Abstract: Monoclonal antibody RR 1/1 directed against the putative LFA-1 ligand molecule intracellular adhesion molecule-1 (ICAM-1) was found to inhibit the T cell proliferative response to the antigen PPD. Interestingly, the percentage of unstimulated monocytes which expressed ICAM-1 on their surface appeared to vary greatly from person to person although the majority of monocytes did express high levels of ICAM-1 within their cytoplasm and surface expression could be rapidly induced on most cells by adherence to fibronectin. Resting T cells showed no evidence of surface or cytoplasmic ICAM-1 although expression was induced both within the cell and on the membrane as a result of activation with phytohemagglutinin or a combination of OKT3 and phorbol 12,13-dibutyrate. The significance of these findings with respect to the function of monocyte and T cell in the generation of an immune response is discussed.

Functional evidence that intercellular adhesion molecule-1 (ICAM-1) is a ligand for LFA-1-dependent adhesion in T cell-mediated cytotoxicity.

Abstract: Although intercellular adhesion molecule-1 (ICAM-1) has been implicated as a ligand in some LFA-1-dependent adhesion, its importance to T cell function has not been established. The present studies investigate the importance of ICAM-1 for human cytotoxic T lymphocytes (CTL), both in their formation of antigen-independent conjugates (AIC) and in their lysis of targets. Analysis of monoclonal antibody (mAb) inhibition of AIC formation indicate that ICAM-1 mAb 1 blocks (a) AIC formation with some but not all targets; (b) the LFA-1 pathway but not the CD2/LFA-3 pathway of adhesion; (c) by binding to the target cell, not the T cell. In studies of cell-mediated lysis (CML) ICAM-1 mAb inhibited lysis of some targets, such as U-937, that use ICAM-1 predominantly in AIC formation; CML on some other targets is not inhibited by ICAM-1 mAb. These data indicate that ICAM-1 is a ligand for AIC formation, antigen-specific CTL recognition and cytolysis of particular target cells. The data also indicate that ICAM-1 is not used in LFA-1-dependent CTL interactions with all kinds of target cells, suggesting the existence of alternative ligands for LFA-1.

Establishment of an interleukin 6 (IL 6)/B cell stimulatory factor 2-dependent cell line and preparation of anti-IL 6 monoclonal antibodies.

Abstract: Murine hybridomas producing monoclonal antibodies (mAb) specific to human interleukin 6 (IL 6/BSF-2) were established. One of these hybridomas (MH60.BSF2) was found to be dependent on IL 6 for its in vitro growth. None of the known biological factors tested, such as recombinant (r) human (Hu) IL 1 alpha, rHuIL 1 beta, rHuIL 2, rHuIL 3, rHuIL 4, rHu interferon (IFN)-gamma, HuIFN-beta, rHuG-CSF, or recombinant murine (Mu) IL 3, MuIL 4, rMuIL 5, could induce the in vitro growth of MH60.BSF2 cells. The half-maximum tritiated thymidine uptake by MH60.BSF2 cells could be achieved by picogram amounts of rIL 6, making this hybridoma clone an indicator cell for specific and sensitive detection of the IL 6 activity in test samples. The MH166.BSF2 clone was found to produce IgG1,chi type mAb (alpha BSF2-166) capable of neutralizing IL 6 activity. The other clone, MH60.BSF2, produced IgM,chi type mAb (alpha BSF2-60) unable to neutralize IL 6 activity. Both mAb specifically reacted with IL 6 as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analysis. An enzyme-linked immunosorbent assay (ELISA) utilizing alpha BSF2-166 and rabbit anti-IL 6 antibodies was established which could detect as low as 50 pg/ml of IL 6. Since both the ELISA and MH60.BSF2 hybridoma could detect small amounts of IL 6 in biological fluids, they constitute powerful tools in exploring the presence or the role of IL 6 in various immunological disorders.

cDNA cloning of murine interleukin-HP1: homology with human interleukin 6.

Abstract: Interleukin-HP1 (HP1) is a murine T cell-derived lymphokine, originally described as a growth factor for B cell hybridomas and plasmacytomas, that was recently shown to stimulate growth and differentiation of normal B and T lymphocytes Here, we describe a cDNA for HP1 that was isolated from a library prepared using mRNA of a murine helper T cell clone activated with a clonotypic antibody The cDNA, which hybridizes with a mRNA of approximately 1300 bp, encodes a polypeptide consisting of 211 amino acids with a typical signal sequence of 24 residues followed by 187 amino acids, which form the mature protein (Mr = 21,710) No N-glycosylation site but several potential O-glycosylation sites were identified in the predicted sequence Comparison of the cDNA sequence of HP1 with that of human interleukin 6 disclosed a homology of 65% at the DNA level and of 42% at the protein level with a maximum of 57% for the segment spanning residues 42-102 of mature HP1 Considering the functional homology that was previously established between these two proteins, we therefore propose that HP1 be renamed murine interleukin 6

Interleukin 6, the third mediator of acute-phase reaction, modulates hepatic protein synthesis in human and mouse. Comparison with interleukin 1 β and tumor necrosis factor-α

Abstract: Interleukin 6 (IL6) is the new definition of a group of cytokines previously named according to their biological activity, e.g. B cell stimulatory factor 2 (BSF-2), hybridoma plasmocytoma-growth factor (HGF), interferon-beta 2 (IFN-beta 2), hepatocyte stimulating factor (HSF). It has recently been suggested that IL6 may represent the major mediator of acute-phase protein response whereas IL1 beta and TNF-alpha could play a minor role. We compared the effect of the three cytokines on hepatic protein synthesis by performing in vitro as well as in vivo experiments. Human hepatoma cells (PLC/PRF5) were exposed to each cytokine separately for 20 h, and the effect was then studied at the protein and RNA level. All three cytokines reduced albumin and increased C3 and ceruloplasmin biosynthesis. The cytokines induced the same effect at the RNA level indicating that the modulation was pretranslational. The effect of the cytokines was specific since actin gene expression was not changed; furthermore the effect was blocked by specific antibodies against the cytokines. The effect of the single cytokines was dose and time dependent, and quantitatively comparable. None of the cytokines was able to alter alpha 1-anti-trypsin synthesis. In vivo experiments with mice showed that IL1 beta and TNF-alpha both induce serum amyloid A (SAA) mRNA in the mouse liver and increase factor B (Bf) gene expression. Human recombinant IL6 induced SAA gene expression and it also had a weak positive effect on Bf gene expression after i.p. injection. These data demonstrate that the three cytokines studied are quantitatively and qualitatively comparable, and that all three are probably involved in acute-phase protein response.

Limiting dilution analysis of proliferative responses in human lymphocyte populations defined by the monoclonal antibody UCHL1: implications for differential CD45 expression in T cell memory formation.

Abstract: Using limiting dilution analysis, we investigated proliferative responses of UCHL1+ and UCHL1− T cell populations to compare the precursor cell frequencies following recall and alloantigen stimulation, and the complexity of cellular interactions within UCHL1+ and UCHL1− populations. We find high frequencies of recall antigen responses among UCHL l+, but not UCHL1− T cells. In contrast, both populations contain similar frequencies of alloantigen responsive cells. Our results are consistent with single-hit kinetics in recall as well as alloantigen responses and show no complex cellular interactions within the responding populations. In conclusion, the difference between the recall antigen response of UCHL 1+ and UCHL1− cells observed in conventional proliferation assays is most probably due to a high frequency of recall antigen-responsive UCHL1+ cells and not to suppressive phenomena in the UCHL1−population. These data suggest that memory T cells are largely of the UCHL 1+ phenotype. The relation of post-thymic T cell maturation and differential CD45 expression is briefly discussed.

Memory B cells in T cell-dependent antibody responses colonize the splenic marginal zones.

Abstract: Specific hapten-binding B cells were identified in the splenic marginal zones following immunization with hapten-protein conjugates. Hapten binding by marginal zone B cells does not appear to be due to passive absorption of anti-hapten antibody. For double immunization with two haptens, 2,4-dinitrophenyl (DNP) and 2-phenyloxazalone (Ox) each conjugated to hemocyanin, resulting in the appearance of discrete DNP-binding cells and Ox-binding cells in the marginal zone. Very few cells were identified which bound both haptens. The hapten-binding cells in the marginal zones have a phenotype characteristic of other marginal zone B cells. They express surface IgM but not IgD. Occasional cells also have surface IgG2c. All hapten-binding cells possessed the antigen recognized by the monoclonal antibody HIS 14 but lacked those identified by HIS24 and HIS22. Hapten-binding B cells were shown to have been in cell cycle shortly before entering the marginal zone but were no longer in cell cycle after arriving at that site. Once in the marginal zone hapten-binding cells were shown to remain in that site for upwards of 2 weeks. Following reimmunization with DNP-hemocyanin, DNP-binding but not Ox-binding cells were lost from the marginal zone. At the same time DNP-binding cells arrived in the periarteriolar lymphocytic sheath and to a lesser extent the follicles. These cells were in active cycle and appeared to give rise both to plasma cells and marginal zone hapten-binding cells. It is concluded that hapten-binding cells found in the marginal zones are memory B cells i.e. they have been derived from B cells which have undergone antigen-driven proliferation, they are no longer in cell cycle but can be induced to re-enter cell cycle by subsequent exposure to antigen. Good antibody responses were obtained following immunization with hapten-polysaccharides; however, no hapten-binding cells appeared in the marginal zones in response to these T cell-independent type 2 antigens.

Interleukin 5 enhances interleukin 4-induced IgE production by normal human B cells. The role of soluble CD23 antigen

Abstract: Interleukin 4 (IL 4)-induced IgE production by peripheral blood lymphocytes and tonsil cells from normal donors was enhanced in a dose-dependent fashion by IL 5. IL 5 tested alone was not effective. The synergistic effects of IL 5 were most pronounced at suboptimal IL 4 concentrations, whereas at saturating IL 4 concentrations (200-300 U/ml), IL 5 had no effect. Interferon-gamma (IFN-gamma) and F(ab')2 fragments of monoclonal antibody 25 directed against the CD23 antigen, that blocked IL 4-induced IgE synthesis, also inhibited the production of IgE in the presence of combinations of IL 4 and IL 5, indicating that IL 5 potentiates the activation pathway through which IL 4 induces IgE production. In contrast, IL 4 (50 U/ml) blocked IL 5-induced IgA synthesis. IL 5 was ineffective in inducing the release of soluble CD23 (sCD23), but in the presence of IL 4 an enhanced release of sCD23 was observed, provided IL 4 was present at suboptimal concentrations. IFN-gamma completely blocked sCD23 release induced by IL 4 and IL 5. These results demonstrate that there is a strong quantitative correlation between sCD23 release and induction of IgE synthesis. sCD23 fraction-correlation between sCD23 release and induction of IgE synthesis. sCD23 fractionated from the Epstein-Barr virus-transformed B cell line RPMI 8866 was ineffective in inducing IgE production. However, sCD23 acted synergistically with suboptimal concentrations of IL 4. sCD23 did not modulate the IgE response at saturating concentrations of IL 4. Collectively, these data indicate that sCD23 plays an important regulatory role in the modulation of IL 4-induced IgE synthesis mediated by IFN-gamma and IL 5.

Action of recombinant human interleukin 6, interleukin 1β and tumor necrosis factor α on the mRNA induction of acute‐phase proteins

Abstract: The rat hepatoma cell line Fao was used to study the role of three inflammatory mediators on the mRNA regulation of several acute-phase proteins. In the presence of 10(-6) M dexamethasone beta-fibrinogen mRNA levels increased 6-fold after addition of recombinant human IL 6 (rhIL 6). rhIL 1 beta or recombinant human tumor necrosis factor alpha (rhTNF alpha) had essentially no effect on beta-fibrinogen mRNA induction but led to a 20-fold increase in alpha 1-acid glycoprotein mRNA in the presence of dexamethasone. On the other hand, rhIL 6 was a much weaker stimulator of alpha 1-acid glycoprotein mRNA synthesis. All three mediators reduced albumin mRNA concentrations to about 30% of controls. Whereas the induction of beta-fibrinogen mRNA was potentiated by dexamethasone, the synthetic glucocorticoid analog was an absolute requirement for the stimulation of alpha 1-acid glycoprotein mRNA. The mRNA levels of the negative acute-phase protein albumin were induced 5-fold by dexamethasone alone. The beta-fibrinogen mRNA induction started immediately after addition of rhIL 6 and reached a maximum between 12 and 18 h. In contrast, the time-course for alpha 1-acid glycoprotein mRNA synthesis showed a lag phase of 8 h followed by an increase up to 20 h after rhIL 1 beta. rhTNF alpha led to an even more delayed increase in alpha 1-acid glycoprotein mRNA. Whereas in the case of beta-fibrinogen mRNA induction no synergistic effect was observed between various concentrations of the three mediators, the combination of rhIL 6/rhIL 1 beta as well as rhIL 6/rhTNF alpha or rhIL 1 beta/rhTNF alpha regulated synergistically alpha 1-acid glycoprotein and albumin mRNA. It is concluded that discrete acute-phase proteins are regulated differently by the inflammatory mediators IL 6, IL 1 beta and TNF alpha, indicating that the acute-phase response is more complex than previously assumed. The Fao cell line used in this study turned out to be an ideal model for acute-phase protein regulation, suitable for the discrimination between the inflammatory mediators IL 6 and IL 1/TNF alpha.

The preferential accumulation of helper-inducer T lymphocytes in inflammatory lesions: evidence for regulation by selective endothelial and homotypic adhesion.

Abstract: The mechanisms which lead to the accumulation of T lymphocytes into inflammatory lesions are not clearly understood. We have previously shown that synovial CD4 T lymphocytes are mostly CDw29+UCHL1+ (helper-inducer cells) and very few carry the CD45R antigen which identifies the suppressor-inducer subset. Synovial CD8+ cells are also CDw29+UCHL1+CD45R-. In the present study, lymphocytes from pleural and peritoneal inflammatory infiltrates were shown to have a similar phenotypic pattern. Furthermore, it was demonstrated that the CDw29+UCHL1+ subset had a greater ability than CD45R+ cells to adhere to endothelial cells and to form homotypic clusters. Differential surface expression of LFA-1 on the two subsets was also shown, but this could not account for the demonstrated adhesion differences. Differences in adhesion between CDw29+/UCHL1+ and CD45R+ cells may explain the preferential accumulation of CDw29+/UCHL1+ cells in inflammatory infiltrates and underlie some of the functional differences between cells taken from sites of chronic inflammation and those from peripheral blood.

Allele and locus‐specific differences in cell surface expression and the association of HLA class I heavy chain with β2‐microglobulin: differential effects of inhibition of glycosylation on class I subunit association

Abstract: The assembly of HLA class I antigens, and the contribution of the single N-linked glycan to this process were examined. We observed a requirement for N-linked glycosylation in the proper assembly and surface expression of HLA-B locus products in particular, although considerable variation was seen within the allelic series of the HLA-A and B loci. We conclude that the single N-linked glycan can contribute in a major way to that conformation of the heavy (H) chain which is competent to associate with the light chain beta 2-microglobulin, and that the presence, rather than the type, of carbohydrate chain is important in this respect. The association of human class I H chains with beta 2-microglobulin shows biphasic kinetics, where an initially rapid phase is followed by a prolonged period during which no further association can be measured. It appears that HLA-C H chains are initially synthesized in amounts similar to HLA-A and B H chains, but associate inefficiently with beta 2-microglobulin, resulting in low expression of HLA-C at the cell surface. The individual stages of assembly and maturation of class I antigens including the transfer from Golgi to cell surface were found to display characteristic allelic variation.

Anti-suppressor effect of cyclophosphamide on the development of spontaneous diabetes in NOD mice.

Abstract: In the NOD mouse, an autoimmune process beginning by 5 weeks of age with lymphocyte infiltration and destruction of insulin-secreting beta cells leads to overt diabetes which begins to appear by 11 weeks of age. Although there is a high incidence of insulitis by 10 weeks of age (greater than 80%) in both males and females, by 30 weeks of age diabetic symptoms have occurred in 53-80% of females and in 12-40% of males. Intraperitoneal injection of a high dose (200 mg/kg) of cyclophosphamide (CY) consistently induces the onset of diabetes in male and female NOD mice at an age when spontaneous diabetes rarely occurs. Spleen T cells from CY-induced diabetic mice are capable of transferring the disease into irradiated nondiabetic syngeneic recipients. This indicates that the diabetogenic effect of CY is not mediated by direct toxicity on pancreatic beta cells but is mediated by abrogation of a suppressor mechanism which may prevent activation of T cells responsible for the development of diabetes in the NOD mouse. Additionally, CY is only effective in NOD mice and not in F1 hybrids between NOD and other strains of mice. Thus, the potential beta cell aggressor mechanism is not present in these hybrids as it is in homozygous mice, which indicates that it is not under the control of dominant genes.

Profiles of lymphokine activities and helper function for IgE in human T cell clones.

Abstract: A large panel of phytohemagglutinin (PHA)-induced T cell clones (690 in total), established from four different human lymphoid tissues (peripheral blood, tonsils, lymph nodes and spleens) by a high-efficiency cloning technique, was characterized according to their pattern of lymphokine production. The majority of both CD4+ and CD8+clones from all lymphoid tissues produced interleukin (IL) 2 and/or interferon (1FN)-γ response to 24-h stimulation with PHA. In contrast, higher proportions of IL4-producing clones were found among CD4+clones from tonsils and spleens than from peripheral blood and lymph nodes, whereas only a minority of CD8+ clones from all lymphoid tissues were found to produce IL4. It was not possible to divide the CD4+ (helper/inducer) clones on the basis of their pattern of lymphokine activity into two clear-cut groups analogous to Thl and Th2 helper clones described in mice. Although 21 out of 503 (4%) CD4+ T cell clones produced IL4, but not IFN-γ or IL2, and 208 (41%) produced IL2 and/or IFN-γ, but not IL4, a total number of 185 (37%) CD4+ clones showed the ability to produce IL4 plus IL2 and/or IFN-γ. All types of CD4+ T cells (as classified according to their pattern of lymphokine activity) provided help for IgG production in allogeneic B cells. In contrast, helper function for IgE was detectable only among the IL4-producing clones. However, the proportion of CD4+ clones providing help for IgE synthesis was significantly lower among those producing IL4 and IFN-γ or IL4, IL2 and IFN-γ than among those producing IL4 alone or IL4 and IL2. Thus, a clear-cut dichotomy between IL4- and IFN-γ-producing Th cells, as found in mice, does not seem to exist in humans. However, IL4 and IFN-γ, even if not always produced by distinct T cell subsets, appear to regulate reciprocally the synthesis of IgE in humans as well.

Mycobacteria-reactive Lyt-2+ T cell lines

Abstract: The biological activities of mycobacteria-reactive Lyt-2+ T cells were characterized in vitro. T cells from mice immunized with killed Mycobacterium tuberculosis or viable M. bovis were restimulated in vitro and cloned under limiting dilution conditions. Several L3T4-Lyt-2+ T cell lines, some of them KJ16+, were established. These T cell lines were capable of lysing mycobacteria-primed macrophages in an antigen-specific way. The cytolytic activity of some T cell lines was found to be class I restricted, whereas others showed antigen-specific killing in the absence of apparent H-2 restriction. Several T cell lines produced interferon-gamma after appropriate stimulation. Furthermore, these T cell lines could induce tuberculostatic macrophage capacities by apparently two different mechanisms, namely by secretion of lymphokines (most probably interferon-gamma) and by direct cell contact. We conclude that CD8 T cells with antigen-specific cytolytic potential are generated during tuberculosis and that these T cells are involved in the immune response to tubercle bacilli.

Differential expression of two t cell receptors, tcr1 and tcr2, on chicken lymphocytes

Abstract: A monoclonal antibody, TcR2, has been shown to recognize an avian homologue of the mammalian alpha/beta T cell receptor (TcR). The TcR2-reactive molecule was found to be a T3-associated heterodimer with relative molecular mass of 90-kDa consisting of disulfide-linked 50-kDa and 40-kDa polypeptides. The sizes of the deglycosylated TcR2 polypeptides differed from those of TcR1, an avian homologue of the mammalian gamma/delta T cell receptor. Immunofluorescence analysis revealed that TcR1 and TcR2 are expressed on separate populations of T cells during their development first in the thymus and then in the periphery. Ontogenetic studies revealed that the TcR1+ thymocytes are generated first and the generation of TcR2+ cells begins approximately 3 days later. While most TcR2+ cells in the thymus expressed both CT4 and CT8, TcR2+ cells in blood and the spleen were either CT4+ or CT8+. The TcR1+ cells in blood and thymus were CT4-CT8-, but the majority of TcR1+ cells in the spleen surprisingly expressed the CT8 marker. The data suggest that TcR1 and TcR2 cells are generated in the thymus as separate T cell sublineages.

B lymphopoiesis on stromal cell clone: Stromal cell clones acting on different stages of B cell differentiation

Abstract: B lymphopoiesis supporting activities of two stromal cell clones, MC3T3-G2/PA6 (PA6) and ST2, were compared. When normal bone marrow cells were cultured in these clones under Whitlock-Witte-type condition, mature B cells were generated only in the culture with the ST2 layer. The cells maintained on the PA6 layer, however, contained the precursor cells giving rise to mature B cells when transferred to the ST2 layer. Thus, PA6 is a stromal cell clone capable of supporting the early B progenitors but cannot support a further maturation step into pre-B cells. The immunoglobulin heavy chain gene configuration of B progenitors maintained on the PA6 layer diversified after their transfer onto ST2 layer. This suggests that they are actually the earliest progenitors. This marked difference in the stromal cell activities between PA6 and ST2 could also be distinguished by stromal cell-dependent pre-B cell lines. Among four ST2-dependent pre-B cell lines tested, two grew only on the ST2 layer, which is capable of supporting B lymphopoiesis, while the others grew both on the ST2 and PA6 layers. These results strongly suggest that the process of intra-marrow B cell development is controlled by more than one signal acting on different stages of B cell differentiation.

Ly-6C is a monocyte/macrophage and endothelial cell differentiation antigen regulated by interferon-gamma

Abstract: Using a new Ly-6C-specific antibody (Monts-1) we show that this class of antigens are differentially expressed on monocytes/macrophages and endothelial cells. Recently elicited peritoneal exudate Mac-1+ mononuclear cells, as well as Mac-1+ mononuclear cells in the bone marrow and in the peripheral blood, express high levels of Ly-6C. Ly-6C+ mononuclear Mac-1+ cells are absent in normal uninflamed skin, but are present in high numbers in skin lesions 3 days after the s.c. injection of lipopolysaccharide, concanavalin A or complete Freund's adjuvant. In addition, large Ly-6C+ mononuclear cells are predominant in chronic granulomas induced by complete Freund's adjuvant. Resident macrophages in a variety of tissues express low levels or in many cases do not express Ly-6C. Two out of three monocyte-like cell lines are Ly-6C+, whereas macrophage-like cell lines are negative. Ly-6C+ monocytes/macrophages lose the Ly-6C antigen within 24 h after in vitro culture. Ly-6C- cultured monocytes and Ly-6C- monocyte-like cell lines, but not fully differentiated macrophages and macrophage-like cell lines, can be induced to express the Ly-6C antigen by interferon-gamma. A population of small vessel endothelial cells in diverse tissues also express high levels of Ly-6C. The present findings suggest that the Ly-6C antigen family, shown by others to be involved in T cell activation, may have more general importance in immune responses and cellular differentiation than previously appreciated.

Signal transduction through cd4 receptors: stimulatory vs. inhibitory activity is regulated by cd4 proximity to the cd31t cell receptor

Abstract: The binding of antibody to the CD4 molecule inhibits mobilization of cytoplasmic free calcium ([Ca2+]i) in response to CD3 cross-linking on resting T cells. Similarly, when CD3 and CD4 are independently and simultaneously cross-linked, calcium mobilization is inhibited when compared to that induced by cross-linking CD3 alone. In contrast, when anti-CD4 and anti-CD3 are cross-linked together, calcium mobilization is substantially higher than from CD3 cross-linking alone. A heteroconjugate consisting of covalently bound CD3 and CD4 monoclonal antibodies (mAb) retains the ability to mobilize [Ca2+]i in CD4 cells at protein concentrations approximately two orders of magnitude lower than the free CD3 mAb, and the activity of the heteroconjugate is inhibitable by free CD4 mAb. The CD3/CD4 heteroconjugate also shows significantly greater activity in stimulation of inositol phosphate IP1, IP2 and IP3 synthesis in T cells than the CD3 mAb alone, and again the activity is inhibited by free CD4 mAb. The activity of the CD3/CD4 heteroconjugate is not simply due to oligomerization, since CD3/CD3 or CD4/CD4 homoconjugates or homoconjugate mixtures did not show increased activity. Other heteroconjugates (CD3/CD5 and CD3/CD28) were not different than the CD3/CD3 homoconjugate in their ability to increase [Ca2+]i. Purified CD4 T cells that do not respond to CD3 mAb in solution do respond to the CD3/CD4 heteroconjugate in solution by proliferating in the presence of a CD28 mAb, with a significant fraction of CD4 cells entering the second cycle within the first three days of stimulation. The CD3/CD4 heteroconjugate co-modulates the CD3 and CD4 receptors, indicating that the heteroconjugate is not simply anchoring the T cell receptor to the T cell surface like anti-CD3 on a solid surface. These results suggest that CD4 plays an active role in signal transduction when brought into close physical proximity to the CD3/T cell receptor complex during major histocompatibility complex class II-restricted antigen presentation.

Stromal cell lines which support lymphocyte growth: characterization, sensitivity to radiation and responsiveness to growth factors.

Abstract: Stromal cells which grow as an adherent layer of Whitlock-Witte cultures are thought to be an essential component of the lymphohemopoietic microenvironment. Stromal cell lines from bone marrow (BM) and spleen have been obtained by treatment of cultures with 5-fluorouracil and selected for their lymphocyte support capacity by measuring the clonal growth of stromal cell-dependent lymphocyte lines in methyl cellulose. Established stromal cell lines differed significantly from stromal cells in primary Whitlock-Witte cultures with respect to expression of certain hemopoietic cell surface markers. For example, the Thy-1 and Mac-3 antigens were expressed by stromal cell lines obtained from BM and spleen, but not by stromal cells in primary cultures. Features common to all stromal cells include synthesis of actins, the neural adhesion molecule N-CAM, and a variety of collagens. Two types of common leukocyte antigens were not significantly expressed. The proliferation and total protein synthetic capacity of lymphocyte-supportive stromal cell lines was sensitive to ionizing radiation. After exposure of the cells to 200 rads, the incorporation of either [3H]thymidine or [3H]Leucine was reduced to less than 50% of control values, but the growth of lymphocytes was augmented in the presence of an irradiated stromal cell layer. The proliferation of stromal cell lines was also affected by exposure to a variety of growth factors. Addition of epidermal growth factor or endothelial cell growth factor augmented BM or spleen-derived stromal cell proliferation, while interferon-gamma had the opposite effect. In general, but not exclusively, lymphocyte growth was inhibited by factors which augmented the proliferation of stromal cells. Novel methods are described for isolating stromal cells and determining their capacity to support lymphocyte growth in vitro. Evidence is presented that this ability is not restricted to BM-derived stromal cells. The function of stromal cells was not dependent on their ability to proliferate, and this may be modulated by immunoregulatory and other growth factors.

Importance of antigen specificity for complement-mediated lysis by monoclonal antibodies.

Abstract: Lysis of human lymphocytes by autologous complement had been studied using a range of monoclonal antibodies against different antigens. Antigen specificity (and not antibody isotype) was the most important factor which influenced cell lysis and this could not be accounted for merely by differences in surface density between antigens. Three antigens with comparable surface density were studied in detail: CAMPATH-1 (lytic), major histocompatibility complex class I (lytic) and leukocyte common antigen (poorly lytic). C1q binding was roughly proportional to antibody binding and dependent on antibody isotype. However, the lytic antibodies were much better able to bind and activate whole C1 than the poorly lytic ones. This result would not have been predicted from traditional concepts of complement activation but can be interpreted in the light of models for C1 activation which involve Fc-Fc interactions, Fc-C1r2s2 interactions and a critical C1q stem-arm angle for C1 binding and activation.

A CD3- subset of CD4-8+ thymocytes: a rapidly cycling intermediate in the generation of CD4+8+ cells.

Abstract: T lymphocytes with the surface phenotype CD4+8- and CD4-8+ are considered to be representative of functionally mature cells. We show here that adult murine thymus contains a subpopulation of CD4-8+ cells that differ from CD4-8+ cells found in the periphery in that they do not express the T cell receptor-associated CD3 molecular complex. Such CD3-4-8+ thymocytes are cortisone sensitive and rapidly cycling in situ. Furthermore, in contrast to mature T cells, most CD3-4-8+ thymocytes express low levels of CD5 and high levels of the B2A2 antigen. CD3-4-8+ thymocytes fail to respond to a variety of mitogenic stimuli in vitro but do give rise upon short-term culture to CD4+8+ cells. It is suggested that CD3-4-8+ thymocytes represent a transitional stage of thymus differentiation between the CD4-8- and CD4+8+ compartments.

Synergistic activation of human T cells by interleukin 1 and interleukin 6

Abstract: Purified human interleukin 6 (IL 6) was found to stimulate the proliferation of human tonsillar and peripheral rosetting T cells subliminally activated with phytohemagglutinin (PHA). This response seemed independent of IL 2 but highly dependent on the presence of accessory cells. Indeed, when accessory cell-depleted tonsillar T cells were activated with PHA and exposed to IL 6, only minimal proliferations were observed. A similar result was obtained with IL 1. However, a combination of these two cytokines induced strong proliferations, indicating that IL 1 and IL 6 plays a synergistic role in the interactions between accessory cells and T lymphocytes.