Interleukin-1beta converting enzyme subfamily inhibitors prevent induction of CD86 molecules by butyrate through a CREB-dependent mechanism in HL60 cells.

To investigate the underlying mechanism for induction of CD86 molecules, we analysed the ability of the histone deacetylase (HDAC) inhibitor, sodium butyrate (NaB), to induce CD86 at the transcriptional level in HL60 cells. Our studies showed that the expression of CD86 on the cell surface was increased by 24 hr of NaB treatment, and the enhancement of CD86 mRNA expression was observed by real-time polymerase chain reaction. When we measured NF-kappaB binding activity, significant activity was induced upon NaB stimulation, which was suppressed by the addition of pyrrolidine dithiocarbamate. Butyrate also induced phosphorylated cAMP response element-binding protein (CREB), which bound to cAMP-responsive elements. Dibutyryl (db) -cAMP induced active CREB and increased the levels of CD86 by 24 hr. These observations indicated that NF-kappaB and/or CREB are crucial for butyrate-dependent activation of CD86 gene expression. We examined the inhibitory effects of various caspase inhibitors on the expression of CD86 in cells treated with NaB, because NaB also induced apoptosis with slow kinetics. Intriguingly, our results demonstrated that inhibitors of the interleukin-1beta converting enzyme subfamily (caspase-1, -4, -5 and -13) blocked the butyrate-induced increase in level of CD86. These inhibitors interfered with CD86 gene transcription in the presence of activated NF-kappaB, whereas phosphorylated CREB was down-regulated in the reactions where these inhibitors were added to inhibit CD86 gene expression. These results suggested that butyrate not only acetylates histones on the CD86 promoter through the suppression of HDAC activity, but that butyrate also regulates CREB-mediated transcription, possibly through the caspase activities triggered by NaB.

Effects of superantigen and lipopolysaccharide on induction of CD80 through apoptosis of human monocytes.

To investigate the mechanisms underlying superantigen (SAg) stimulation, we analyzed the effect of SAg on monocyte responses with or without lipopolysaccharide (LPS). Addition of gamma interferon (IFN-gamma) to unstimulated cultures induced a marked increase in the number of CD80(+) monocytes, which was inhibited by LPS through the action of interleukin-10. However, CD80(+) monocytes began to increase before IFN-gamma production, observed after 9 h of stimulation with staphylococcal enterotoxin B (SEB). SEB selectively increased the number of apoptotic CD80(-) monocytes, whereas LPS-treated monocytes were resistant to the apoptotic action of SEB. This SEB-induced killing was abrogated by anti-CD95 monoclonal antibody (MAb) ZB4 and anti-CD95 ligand (CD95L) MAb NOK2, suggesting a CD95-based pathway of apoptosis. Furthermore, the numbers of SEB-induced CD80(+) monocytes were partially decreased by anti-CD119 (IFN-gamma receptor) MAb and by anti-CD95L (NOK2) MAb. The CD30 expression of CD27(high) T cells induced by SEB was increased by agonistic anti-CD95 (CH11) MAb. Together, our findings showed that SEB-induced monocyte apoptosis is closely associated with the enrichment of CD80(+) monocytes generated before IFN-gamma production, followed by up-regulation of CD80 by IFN-gamma, and that LPS has negative effects in both cases. These results also suggested that induction of monocyte apoptosis is an important mechanism by which SAg exerts its anti-inflammatory effects.

Lipopolysaccharide-dependent down-regulation of CD27 expression on T cells activated with superantigen.

To investigate the mechanisms underlying T-cell responses during superantigen (SAg) stimulation, we analysed the effects of SAg on CD27 expression with or without lipopolysaccharide (LPS) as a novel regulator of T-cell function. CD27 is expressed on the majority of resting peripheral blood T cells (CD27low). Activation of T cells by SAg induces high levels of CD27 surface expression (CD27high) accompanied with simultaneous CD30 receptor expression. After prolonged activation in vitro, the level of CD27 expression became intermediate. The effects of LPS on down-regulation of CD27high expression on CD30+ T cells were dose-dependent. Separating LPS-stimulated monocytes from T cells by mechanical dispersion abolished its inhibitory effect, indicating the requirement for direct interactions between monocytes and T cells. We also found that SAg up-regulated CD80 expression on CD14+ monocytes and LPS inhibited SAg-induced CD80 expression after 24 hr of stimulation. Up-regulation of CD152 (CTLA-4) was selective, since it was found to be preferentially expressed on the CD30+ population. Competitive experiments using soluble blocking peptides showed that addition of CD28 or CD80 peptide recovered LPS-induced down-regulation of CD27high expression on CD30+ T cells. These observations suggested that the presence of low levels of CD80 on monocytes may partially inhibit CD27 expression due to inefficient delivery of positive signals via CD28/CD80 interaction, and that the increased levels of CD80 enhance the inhibition through interactions with CD152 which is expressed at the highest levels after 48 hr of activation.

Lipoteichoic acid acts as an antagonist and an agonist of lipopolysaccharide on human gingival fibroblasts and monocytes in a CD14-dependent manner.

CD14 has been implicated as a receptor of lipoteichoic acid (LTA) and other bacterial components as well as lipopolysaccharide (LPS). Since the structures of LTAs from various gram-positive bacteria are heterogeneous, we analyzed the effects of LTAs on the secretion of interleukin-8 (IL-8) by high- and low-CD14-expressing (CD14(high) and CD14(low)) human gingival fibroblasts (HGF). While Bacillus subtilis LTA had an IL-8-inducing effect on CD14(high) HGF which was considerably weaker than that of LPS, Streptococcus sanguis and Streptococcus mutans LTAs had practically no effect on the cells. B. subtilis LTA had only a weak effect on CD14(low) HGF, as did LPS. S. sanguis and S. mutans LTAs at a 1,000-fold excess each completely inhibited the IL-8-inducing activities of both LPS and a synthetic lipid A on CD14(high) HGF. The effect of LPS was also inhibited by the presence of an LPS antagonist, synthetic lipid A precursor IVA (LA-14-PP), with a 100-fold higher potency than S. sanguis and S. mutans LTAs and by anti-CD14 monoclonal antibody (MAb). S. sanguis and S. mutans LTAs, LA-14-PP, and anti-CD14 MAb had no significant effect on phorbol myristate acetate-stimulated IL-8 secretion by HGF. These LTAs also inhibited the IL-8-inducing activity of B. subtilis LTA on CD14(high) HGF, as did LA-14-PP and anti-CD14 MAb. The antagonistic and agonistic functions of LTAs were also observed with human monocytes. Binding of fluorolabeled LPS to human monocytes was inhibited by S. sanguis LTA, although the inhibition was 100 times weaker than that of LPS itself, and anti-CD14 MAb inhibited fluorolabeled LPS and S. sanguis LTA binding. Binding of LTAs to CD14 was also observed with nondenaturing polyacrylamide gel electrophoresis. These results indicate that LTAs act as antagonists or agonists via a CD14-dependent mechanism, probably due to the heterogeneous structure of LTAs, and that an antagonistic LTA might be a useful agent for suppressing the periodontal disease caused by gram-negative bacteria.

Variation in CD4+ T and CD8+ T lymphocyte subpopulations in bovine mammary gland secretions during lactating and non-lactating periods.

Mammary gland secretions (MGS) of dairy cows at different stages of lactation were studied by immunofluorescence cytometry for T lymphocyte subpopulations using monoclonal antibodies. During early and late lactation, the mean ratio of CD4+/CD8+ T lymphocytes in the MGS was 0.5 and 0.8, respectively. A large proportion of the CD8+ cells coexpressed the activation molecule, ACT2. These results indicate that CD8+ ACT2+ cells constituted the major phenotype in the T lymphocytes throughout lactation. In the mammary gland of cows in which drying off was induced, however, the proportion of CD8+ ACT2+ cells decreased, resulting in the increase of the CD4+/ CD8+ ratio in the MGS. At the late non-lactation stage, the ratio reached a maximal level of 2.5-4.0, which was similar to or higher than that found in the peripheral blood. This selective increase of CD4+/CD8+ cell ratio correlated with an increase in the concentrations of total cells in the MGS. This high CD4+/CD8+ cell ratio during the drying off stage rapidly decreased just before parturition, correlating with the decrease in concentrations of total cells in the MGS, reaching the lowest level at early lactation. The cells isolated at the non-lactation stage produced the cytokines IL-2 and IL-4 at a level much higher than those of cells isolated at lactation stages, and the increases were correlated with the CD4+ T lymphocyte proportions.

Heterogeneous expression and release of CD14 by human gingival fibroblasts: characterization and CD14-mediated interleukin-8 secretion in response to lipopolysaccharide.

To identify the role in periodontal inflammatory diseases of human gingival fibroblasts (HGF), the major constituents of gingival tissue, the expression of CD14, a possible lipopolysaccharide (LPS) receptor, and the release of soluble CD14 (sCD14) by HGF were examined. Among the HGF samples from the nine donors tested, more than 50% of the HGF from five donors expressed CD14 but less than 20% of HGF from the other four donors did so, as determined by flow cytometric analysis. The CD14 expression on the cell surface was correlated with the expression of CD14 mRNA. The HGF and skin and lung fibroblasts tested expressed no CD18, which indicates that fibroblasts do not possess other LPS receptors, such as CD11b/CD18 and CD11c/CD18. The CD14 expression by the HGF was decreased after subculturing and was highest at the confluent stage of culture. The treatment of high-CD14-expressing (CD14(high)) HGF with phosphatidylinositol-phospholipase C reduced CD14 expression; this result and the increase in a 55-kDa CD14 indicate that the membrane CD14 (mCD14) on the HGF may be a 55-kDa glycosylphosphatidylinositol-anchored protein. CD14(high) HGF spontaneously released 48- and 57-kDa sCD14. The total release of sCD14 by the HGF was augmented by gamma interferon and Escherichia coli LPS in accordance with the increased expression of mCD14. The CD14(high) HGF secreted interleukin-8 in response to LPS, and the secretion was completely inhibited by anti-CD14 antibody. These results suggest that (i) HGF consist of populations that are heterogeneous on the basis of different levels of expression of CD14 and (ii) CD14(high) HGF secrete inflammatory cytokines in response to LPS via CD14.

Peripheral blood lymphocytes from psoriatic patients are hyporesponsive to beta-streptococcal superantigens.

The strong association of acute guttate psoriasis and streptococcal throat infection, together with the preferential use of T cells expressing a particular T-cell receptor, has suggested a role for bacterial superantigens in the pathogenesis of psoriasis. We examined the proliferative responses of peripheral blood lymphocytes (PBLs), obtained from patients with psoriasis and from healthy controls, to streptococcal superantigens, cytoplasmic membrane-associated protein (CAP) and secretion-type CAP (SCAP), isolated from group A, beta-haemolytic streptococci. PBLs from patients with psoriasis showed significantly less response to SCAP and CAP than those from healthy controls. Because there was no difference between psoriatic patients and controls in the proliferative response of PBLs to staphylococcal enterotoxin A or E (SEA, SEE) or the mitogen phytohaemagglutinin (PHA), these findings strongly suggest that the reduced reactivity to the streptococcal superantigens seems to reflect anergy of a population of PBLs to the superantigens. As the CAP used in the present study stimulates V beta 8 T cells selectively, we further examined the proliferation of V beta 8 T cells after such stimulation using flow cytometry. V beta 8 T cells obtained from three of four psoriatic patients failed to proliferate in the presence of CAP, whereas they proliferated vigorously in the presence of SEE, which activates V beta 8 T cells, confirming the specific hyporesponsiveness of PBLs from psoriatic patients to streptococcal superantigens. We then determined the effects of serum factors on the suppressed response of PBLs to the streptococcal superantigens with SCAP or CAP. It was partially restored when PBLs were cultured with sera obtained from healthy subjects, although the responses were still significantly lower than those of the healthy controls. In contrast, psoriatic sera markedly suppressed the proliferative response of PBLs from healthy controls to CAP or SCAP, but showed no suppression of the proliferative response of PBLs to SEA. Because these findings suggest the presence of specific inhibitory factors in psoriatic sera, we examined whether the inhibitory effect was caused by antisuperantigen antibody. However, no significant increase was detected in antibody titre to CAP in psoriatic sera, as has been noted in sera from patients with poststreptococcal glomerulonephritis. The present results show for the first time the hyporesponsiveness of PBLs to streptococcal superantigens and the presence of serum inhibitors that specifically inhibit T-cell response to the superantigens in psoriatic patients. These findings suggest a pathological role for streptococcal infections in the pathogenesis of psoriasis.

Superantigenicity of helper T-cell mitogen (SPM-2) isolated from culture supernatants of Streptococcus pyogenes.

A superantigen (Streptococcus pyogenes mitogen-2; SPM-2) that stimulates human helper T cells bearing unique types of variable domains of T-cell receptor beta-chain (TCR V beta) was isolated from the culture supernatant of S. pyogenes strain T12. The active molecule isolated by diethylaminoethyl (DEAE)-cellulose chromatography and isoelectric focusing was a protein with a molecular weight (MW) of 29,000 and isoelectric point (pl) of 6.0. This new superantigen was found to activate preferentially V beta 4+, 7+, and 8+ T cells, whereas recombinant streptococcal pyrogenic exotoxin A and C activated V beta 12+ and V beta 2+ T cells, respectively, as determined by flow cytometry and reverse transcriptase-polymerase chain reaction (RT-PCR) methods. This proliferative response was significantly inhibited by anti-HLA-DR monoclonal antibody, and required paraformaldehyde-fixed antigen-presenting cells (APC), indicating that this action is dependent on major histocompatibility complex (MHC) class II molecules without processing. Analysis of the amino-terminal amino acid sequence of the molecule failed to find any identical or significantly homologous proteins. We have previously reported that cytoplasmic membrane-associated protein (CAP), a streptococcal superantigen isolated from the cell membranes of S. pyogenes T12 strain, stimulated mainly V beta 8+ T cells. Both SPM-2 and CAP preferentially stimulated helper T cells, and rabbit antiserum against SPM-2 completely neutralized the T-cell-stimulating activities of CAP, suggesting that SPM-2 and CAP belong to a family of streptococcal mitogenic proteins. The SPM-2 activity with stimulation of V beta 8+ T cells was detected extensively in the culture fluids of group A streptococci, but not in those of other streptococcal species, including groups B and D streptococci, and most of the activities detected were completely inhibited by anti-SPM-2 serum. These results indicate that SPM-2 may be a newly discovered superantigen molecule, which can be commonly synthesized by group A streptococci.

Accessory cell function of a human colonic epithelial cell line HT-29 for bacterial superantigens.

The expression and up-regulation of cell adhesion molecules on a human colonic epithelial cell line HT-29, and the peripheral blood T lymphocyte proliferation responses to bacterial superantigens presented by this cell line were investigated, compared with peripheral blood monocytes. In HT-29 cells, there was constitutive expression of intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-3 (LFA-3) at a low level, but no constitutive expression of HLA-DR, LFA-1, B7-1 and B7-2 molecules. After stimulation with the supernatants of staphylococcal enterotoxin B (SEB)-stimulated peripheral blood mononuclear cells for 48 h, there was significant up-regulation of HLA-DR and ICAM-1 molecules (both > 90% positive). However, this stimulation had no effect on the expression of LFA-1, B7-1, B7-2 and LFA-3 molecules. In the presence of all tested superantigens SEB, toxic shock syndrome toxin-1, and streptococcal pyogenic exotoxin A, stimulated HT-29 cells caused significant T cell proliferation. When monocytes were used as antigen-presenting cells (APC), the MoAbs against HLA-DR, B7-2 and LFA-3 showed a significant inhibition of SEB-induced T cell proliferation. Anti-ICAM-1 MoAb had no effect on this response. On the other hand, when stimulated HT-29 cells were used as APC, the MoAbs against HLA-DR and ICAM-1 significantly inhibited SEB-induced T cell proliferation. In contrast to monocytes, anti-B7-2 and anti-LFA-3 had no effect on this response. SEB could not induce HT-29 cells to produce IL-8 directly; however, SEB significantly induced the stimulated HT-29 cells to produce IL-8 in the presence of T cells. Thus these data demonstrate that the products of superantigen-stimulated T cell activation can increase the expression of HLA-DR and ICAM-1 molecules on HT-29 cells significantly. Stimulated HT-29 cells can serve as APC to bacterial superantigens. This response is an HLA-DR- and ICAM-1-dependent, but B7-2- and LFA-3-independent process, which was different from professional APC monocytes.

Isolation of a new superantigen with potent mitogenic activity to murine T cells from Streptococcus pyogenes.

A mitogenic substance on murine lymphocytes was detected in the culture supernate of Streptococcus pyogenes type 12 strain. This substance had a molecular weight of 28,000 and pI 9.2, and was designated as S. pyogenes mitogen (SPM). The proliferative response of C3H/HeN spleen cells began at 1 ng ml-1 and reached a maximal response at 100 ng ml-1 of SPM for 4 days culture. Anti-Thy 1.2 mAb and complement-treated spleen cells abrogated the proliferative response to any dose of SPM. Although the anti-major histocompatibility complex class 1 mAbs had no blocking effect on proliferation by SPM, this proliferation was substantially inhibited by the addition of either anti-I-A or anti-I-E mAb, and complete inhibition was produced by the addition of both mAbs. Fixed antigen-presenting cells still induced T cell proliferation by SPM. A significant expansion of T cells bearing V beta 13 T-cell receptor was observed up to 73% among the Thy 1.2+ cells in cultures stimulated with SPM, indicating expansion in a V beta-specific manner. Immunoblotting of IEF-separated proteins showed that anti-streptococcal pyrogenic exotoxin (SPE) C reacted with a protein of pI 6.9 and anti-SPEB did not show any reactivity. SPEA was reported to expand V beta 8.1 and 8.2 bearing murine T cells, and SPM did not. SPM also exhibited potent mitogenic activity on human T cells and V beta 21+ T cells were selectively expanded. These results lead to the conclusion that SPM was neither SPEA, B nor C, but a new protein belonging to a group of streptococcal superantigens with activity on not only human but also murine lymphocytes.

Adhesion molecules on intermediate TCR cells. II. Hepatoprotective effects of hyaluronic acid on acute liver injury.

The liver is a major organ wherein extrathymic T cells and NK cells exist in mice. Due to their unique properties, i.e., extrathymic T cells are TCR (or CD3)-intermediate+ IL-2R beta+ (herein termed intermediate TCR cells) and NK cells are TCR(-)IL-2R beta+, they are easily distinguished from the other lymphocyte subsets by using mAbs in conjunction with immunofluorescence tests. They were recently found to express a higher level of CD44 antigen, which is a ligand for hyaluronic acid, than that of another T cell subset (i.e., thymus-derived T cells or bright TCR cells). Since an intravenous administration of hyaluronic acid was also found to reduce the number of intermediate TCR cells and NK cells in the liver, we examined whether hyaluronic acid had a hepatoprotective effect on acute liver injury. Such injury was induced by LPS injection in mice pretreated with Propionibacterium acnes 1 week earlier. When a single dose of hyaluronic acid was given to these mice 12 hr before LPS injection, a prominent hepatoprotective effect was observed in terms of decreases of mortality (up to 50%), lymphocyte infiltration of the liver, serum transaminase levels, and tissue damage. At this time, liver mononuclear cells isolated from the treated mice showed decreased levels of cytokine production such as TNF and IL-1. These results reveal that intermediate TCR cells and NK cells in the liver actually adhere the sinusoid walls by means of an interaction of CD44 molecules and hyaluronic acid even in the case of acute liver injury. It suggests a possible therapeutic effect of the administration of hyaluronic acid in acute liver injury by eliminating the effector cells and cytokine-producing cells from the liver.

Cytoplasmic membrane-associated protein (CAP) isolated from Streptococcus pyogenes: as a new bacterial superantigen.

A protein isolated from the cytoplasmic membranes of Streptococcus pyogenes (cytoplasmic membrane-associated protein, CAP) stimulated human T cells in vitro to induce their mitogenic response. This CAP-induced T cell proliferation required the presence of nylon-adherent accessory cells (AC) of either autologous or allogeneic origin in the reaction mixtures. In addition, the reaction was inhibited by monoclonal antibodies (mAbs) against major histocompatibility complex (MHC) class II molecules, HLA-DR and -DQ, but not -DP. Human lymphoid cell lines positive for HLA-DR but not those lacking it were also effective as AC for the reaction. A binding test using fluorescein-labeled protein revealed that CAP bound to the adherent monocytes and HLA-DR+ but not to -DR- lymphoid cell lines. The proliferative response of T cells to CAP was, however, not inhibited by the addition of the lysosomotrophic agent NH4Cl to the reaction mixtures. These results suggest that the presentation of CAP by AC to human T cells is mediated through binding of the protein to the MHC class II molecules but without being processed in the AC. The proliferative response of T cells was also found to be inhibited by addition of anti-CD2, -CD3 or -T cell receptor (TcR) mAbs. A major population responding to CAP was CD3+4+8- T cells. CAP also appears to stimulate T cells bearing V beta 8 sequences much more selectively than T cells bearing other V beta s. These results indicate that this streptococcal membrane protein, CAP, may be a new protein belonging to a group of bacterial superantigens.

[The roles of cytokine in organ-specific tumor metastasis].

The production of cytokines by tumor cells has been suggested as the molecular perturbation responsible for the development of malignant tumors. The behavior of tumor cells in animals is presumed to be affected by these factors, which include such hematopoietic cytokines as GM-CSF, IL-1, and IL-6. Here, we report findings demonstrating that GM-CSF is produced by many murine transplantable tumors with metastatic ability in the lungs, and IL-6 and/or IL-1 are produced by the tumors metastatic in the liver. We discuss the notion that the particular organ or organs in which tumor cells metastasize may be associated with the type of cytokines produced by the tumors. Metastatic spread requires interactions of tumor cells with components of the extracellular matrix of host tissues or with other cells, almost all of which depend on cell surface determinants such as cell adhesion molecules. We also discuss the possibility that the expression and adhesive potentials of adhesive protein (CD44) may be regulated by the cytokine (GM-CSF) excreted from the tumor cells. We then emphasize the possibility that both gene expression of cytokines and adhesive proteins play a critical role in tumor metastasis and in determining organ specificity in metastasis.

Induction of acute arthritis in mice by peptidoglycan derived from gram-positive bacteria and its possible role in cytokine production.

The activities of a water-soluble peptidoglycan fragment derived from Staphylococcus epidermidis (SEPS) were examined as to their role in proliferation of spleen mononuclear cells (SMNC) from various strains of mice, the production of cytokines in vitro, and the induction of an inflammatory reaction in vivo. The proliferation of SMNC from C3H/HeN, C57BL/6, AKR, DBA/2, and ddY mice in reaction to SEPS in vitro showed a peak on day 3 and was greater than that of SMNC from BALB/c mice. The cells of SMNC from C3H/HeN mice responsive to SEPS were indicated to be mainly macrophages. A time kinetics experiment showed a coincidence in the proliferation of SMNC in reaction to SEPS and the detection of colony-stimulating factor (CSF) activity. Interleukin 2 (IL-2) activity was not detected during the incubation periods. When SEPS was administered to mice, much stronger mRNA transcripts of granulocyte-macrophage (GM)-CSF were detected in the lungs of C3H/HeN mice than in BALB/c mice. On the other hand, the amounts of IL-1 and PGE2 produced by SMNC of BALB/c mice stimulated by SEPS were greater than those produced in C3H/HeN mice. SEPS was confirmed to induce arthritis in BALB/c mice, but not in C3H/HeN mice. Our findings suggest that the production of GM-CSF is involved in the in vitro proliferation of SMNC in reaction to SEPS and that along with IL-1 and PGE2 production, contributes to the inflammation by SEPS in vivo.

Mechanism of stimulation of T cells by Streptococcus pyogenes: isolation of a major mitogenic factor, cytoplasmic membrane-associated protein.

Our previous studies established that heat-killed Streptococcus pyogenes, as well as other gram-positive cocci, when incubated with human peripheral blood leukocytes (PBLs) in culture, induced polyclonal activation of T lymphocytes. The activated T lymphocytes included CD4+ CD8- helper T cells and CD3+ and CD4- CD8- double-negative T cells with gamma delta T-cell receptors. In the present study, we isolated a major factor with this unique mitogenic activity against human T lymphocytes from S. pyogenes. This active fraction was found in the cytoplasmic membrane (CM) of the heat-killed organisms but not in other cellular fractions such as cell walls, peptidoglycan, lipoteichoic acids, or cytoplasmic soluble fractions. An active molecule(s) was further isolated from the CM by cholic acid extraction followed by Sephacryl S-200 chromatography. The molecule was protease labile but highly resistant to heat, had a pI of greater than or equal to 9.3 and a molecular weight of 10,000 to 15,000 according to gel filtration experiments, and was termed CM-associated protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the protein purified by anion-exchange chromatography showed a single band with a molecular weight of 15,000, corresponding to mitogenically active regions. Purified CM-associated protein induced activation of T lymphocytes, which consisted of CD4+ CD8- T cells and CD4- CD8- double-negative T-cell receptor gamma/delta + T-cell populations, as did the whole cells of S. pyogenes.

GM-CSF and G-CSF stimulate the synthesis of histamine and putrescine in the hematopoietic organs in vivo.

Histamine and putrescine (a precursor of polyamines) are formed by histidine decarboxylase (HDC) and ornithine decarboxylase (ODC), respectively. Within a few hours after injection of a lipopolysaccharide (LPS) into mice, HDC is induced in the liver, spleen, lung and bone marrow, and ODC is induced in the liver, spleen and bone marrow. Since LPS is known to stimulate the production of various cytokines, the abilities of various cytokines to induce HDC and ODC in the tissues of mice were examined. IL-2, IL-6, IL-8, IFN gamma and M-CSF were ineffective. IL-1 alpha, IL-1 beta, TNF alpha and TNF beta induced HDC and ODC, as does LPS. On the other hand, GM-CSF and G-CSF induced HDC and ODC only in the spleen and bone marrow within a few hours after their injection. These results suggest that, in addition to their roles in inflammation or immune responses, HDC and ODC are also involved in an early stage of hematopoiesis.

Activation of extrathymic T cells in the liver and reciprocal inactivation of intrathymic T cells by bacterial stimulation.

We recently demonstrated that the liver might be a major site of extrathymic T cell differentiation, including both alpha beta and gamma delta T cells. This extrathymic pathway in the liver, which has a relatively minor role in normal young mice, is activated in mice under bacterial stimulation. In the present study, we investigated how the extrathymic and intrathymic T cell differentiations were mutually related in mice injected intravenously with 10(8) heat-killed Escherichia coli. Three days after stimulation, extrathymic T cells in the liver were observed to be prominently activated in terms of increases in the total number of cells yielded, spontaneous cell proliferation in in vitro culture, and intermediate alpha beta TCR cells. Intermediate alpha beta TCR cells were extrathymic T cells uniquely seen in the liver. However, at the same time intrathymic T cells were profoundly inactivated, showing decreases in the number of thymocytes (more than 90% atrophy), spontaneous cell proliferation, and dull TCR cells with double positive CD4+8+ phenotype. With time, these responses were reversed and normal states were regained. These results suggested that extrathymic and intrathymic T cells are always activated or inactivated in the opposite direction, and that the liver and the thymus are dynamic immune organs. It raises the possibility that the extrathymic T cell differentiation in the liver and the intrathymic T cell differentiation may be reciprocally regulated by certain factors.

Increased in vivo production of tumor necrosis factor after development of diabetes in nontreated, long-term diabetic BB rats.

We have recently reported that chronic and systemic administration of tumor necrosis factor alpha (TNF) inhibits development of autoimmune diabetes in NOD mice and BB rats, animal models of insulin-dependent diabetes mellitus (IDDM). During these experiments, we unexpectedly found that in vivo production of TNF stimulated by a single injection of lipopolysaccharide was enhanced approximately 10 times in the long-term diabetic BB rats (P less than 0.0001), whose mean duration of diabetes with more than 16.8 mM (300 mg/dl) of nonfasting blood glucose level was 26.2 +/- 2.1 days, as compared to that in the rats of nondiabetes and in the rats at the onset of diabetes, whose mean duration of diabetes was 1.4 +/- 0.6 days. The long-term diabetic, but not short-term-diabetic, rats were also associated with increased levels of serum fructosamine/albumin (P less than 0.01) and triglyceride (P less than 0.01) and with a decreased level of serum albumin (P less than 0.01). The in vivo TNF productivity in the diabetic rats, including the short-term- and long-term-diabetic rats, was correlated positively with the level of fructosamine/albumin (P less than 0.05) and negatively with the level of serum albumin (P less than 0.05), but not with levels of blood glucose. None of these correlations were observed in nondiabetic rats. The increased LPS-induced serum TNF activity in the long-term diabetic state was observed not only in BB rats but also in NOD mice and GK rats, a model of non-IDDM, irrespective of sexes and ages, indicating that the enhancement of in vivo TNF production was a result of long-term diabetes. These findings indicate that some factor(s) associated with the long-term-diabetic state may prime macrophages in vivo to produce TNF. Further study is needed to reveal a mechanism of the enhanced TNF production and its possible relevance to various abnormalities associated with the chronic hyperglycemic state.

Polyclonal activation of human lymphocytes and induction of cytotoxic lymphocytes by streptococcal preparations.

Polyclonal activation of human peripheral blood lymphocytes (PBLs) in vitro by preparations of Streptococcus pyogenes Su strain (OK-432) and other heat-killed strains was investigated. The streptococcal preparations tested induce a proliferative response of PBLs via interleukin-2 (IL-2)-independent pathways. The proliferative response is accompanied by the generation of lymphoblastic cells (LBCs), which consist of heterologous lymphocyte populations: CD4+ helper type of T cells, and CD4-CD8- double-negative (DN) lymphocytes, including both CD3+ TcR gamma + T cells and CD2+CD3- immature type of T or non-T cell type of lymphocytes. Almost all the LBCs express Leu19, TfR (transferrin receptor), LFA-1 and CD38 (OKT10) antigens, which are expressed on activated T cells, NK cells and some other lymphocytes. The proliferative response of human PBLs is also accompanied by the generation of potent cytotoxic activity against NK-sensitive and -resistant targets. C-dependent cytolysis and cell sorting experiments of OK-432-activated LBCs revealed that both CD3+ and CD3- types of CD4-CD8- DN lymphocytes, but not CD4+ helper T cells, may be major populations responsible for the cytotoxicity induced. On the other hand, CD4-CD8- T cells may be required for the proliferation of PBLs and generation of cytotoxic effector cells. These results suggest that the OK-432 and other streptococcal preparations stimulate the human PBLs in vitro to induce the proliferation/activation of CD4+ T cells, mediating the following generation of DN cytotoxic effector lymphocytes.