Differential effects of human immunodeficiency virus isolates on beta-chemokine and gamma interferon production and on cell proliferation

All human immunodeficiency virus (HIV) isolates can grow readily in primary CD4(+) T cells, but they can be distinguished by their ability to replicate in macrophages and established T-cell lines. The macrophage-tropic viruses are generally non-syncytium inducing (NSI), whereas the T-cell-line-tropic viruses are syncytium inducing (SI) in cultured cells. We now demonstrate that infection of CD4(+) T cells by NSI and SI viruses shows a differential effect on production of beta-chemokines and gamma interferon. Infection by NSI viruses increased production of MIP-1alpha, MIP-1beta, and gamma interferon, whereas infection by SI viruses had no effect or decreased production of these cytokines. Production of RANTES was slightly increased during infection by both virus phenotypes. This differential effect of NSI and SI viruses was observed at the level of beta-chemokine mRNA as well as at the level of protein expression. Infection by NSI viruses also increased CD4(+) cell proliferation. These results may have relevance for a differential role of HIV strains in AIDS pathogenesis.     

Role of liver NK cells and peritoneal macrophages in gamma interferon and interleukin-10 production in experimental bacterial peritonitis in mice

Gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and interleukin-10 (IL-10) production by liver, spleen, lung, peripheral blood mononuclear cells (MNC), and peritoneal exudate cells (PEC) in experimental bacterial peritonitis was examined by cecum ligation and puncture (CLP) (with an 18-gauge needle) of BALB/c mice. MNC of organs were cultured for 18 h, and cytokine levels in supernatants were examined. Cytokines contained in peritoneal lavage fluid were regarded as those produced by PEC. Only liver MNC and PEC produced substantial amounts of IFN-gamma, and PEC were the main source of IL-10, especially 12 h after CLP. As reflected by the cytokine production by liver MNC and PEC, serum IFN-gamma and IL-10 levels were elevated after CLP. C57BL/6 (B6) mice and BALB/c nude mice showed a similar pattern of cytokine production. TNF-alpha levels in culture supernatants, peritoneal lavage fluid, and sera were not significantly elevated compared to those of sham-operated mice. In vivo depletion of NK cells of B6 mice with anti-asialo GM1 or anti-NK1.1 antibody greatly decreased IFN-gamma levels in liver MNC culture supernatants and sera, suggesting that liver NK cells are IFN-gamma producers. On the other hand, plastic-adherent PEC macrophages are the major IL-10 producers. Mice subjected to a cecum ligation and cut procedure (which have a more severe peritonitis) showed much higher IFN-gamma and IL-10 levels than those subjected to CLP, while mice subjected to CLP with a smaller (22-gauge) needle showed low levels of these cytokines. These findings show that liver NK cells and PEC macrophages are important for the production of proinflammatory and anti-inflammatory cytokines in bacterial peritonitis.     

Expression of costimulatory molecules, B7-1 and B7-2 on human gastric carcinoma

Costimulation of T cells via B7-1 and B7-2 molecules on a tumor has been shown to be important for eliciting cell-mediated antitumor immunity. We studied the surface expression of B7-1 and B7-2 in 24 cases of gastric carcinoma from the primary locus, 20 cases of metastatic carcinoma from malignant ascites, 20 cases of benign gastric mucosa and 7 gastric carcinoma cell lines by two-color flow cytometry with mAb CD80 and CD86. The B7-1 and B7-2 molecules were expressed by 6 cell lines, and 1 cell line showed the predominant expression of B7-2 but not B7-1. Almost all patients with primary gastric carcinoma and benign gastric mucosa showed high levels of expression of the B7-1 and B7-2, revealing approximately 40%-60% positive cells. However, the percentage of B7-1-positive cells of poorly differentiated primary carcinomas was significantly lower than that of well-differentiated carcinoma and normal mucosa (P < 0.01). Furthermore, all of the metastatic carcinoma cells revealed consistently very low or undetectable levels of expression of the B7-1 molecule, only 8% (mean) of cells being positive, despite showing higher levels of B7-2 expression. Thus, it seems likely that decreased or deleted expression of B7-1 correlates with the grade of tumor differentiation, tumor progression and metastasis. These results suggest that the B7-1 molecule on the gastric carcinoma bearing CD80+CD86+ is abrogated during tumor invasion and/or metastasis, and the tumor finally acquires the CD80-CD86+ phenotype. Consequently, inadequate B7-1 costimulation may contribute to the escape of tumors from destruction by the host's immune system.     

Antigen provoking gamma interferon production in response to Mycobacterium bovis BCG and functional difference in T-cell responses to this antigen between viable and killed BCG-immunized mice

It has been shown that gamma interferon (IFN-gamma)-producing CD4+ T cells, which are generated only by immunization with viable bacteria, exert a significant role in protective immunity against mycobacteria in mice. In this study, we have tried to determine the antigen recognized by the T cells in search of a possible protective antigen. T cells from viable Mycobacterium bovis BCG-immunized mice were stimulated with several antigens, and IFN-gamma production was measured. Purified protein derivative and viable and killed BCG lysates caused significant IFN-gamma production, and almost the same level of IFN-gamma activity was detected in both groups stimulated with viable and killed BCG lysates. However, heat shock protein (HSP) 65 and HSP 70 were not a major antigen for IFN-gamma production. The antigen provoking IFN-gamma production is localized mainly in the membrane fraction of BCG cells, and the approximate molecular size was 18 kDa. On the other hand, T cells from killed BCG-immunized mice never responded to this antigen for IFN-gamma production, whereas they could mount a delayed-type hypersensitivity response. These results showed that the antigen provoking IFN-gamma production was present in killed as well as viable BCG. In addition to the antigen presentation by antigen-presenting cells, some kinds of differentiation factor (such as monokines) that are produced only by stimulation with viable cells seemed to be necessary for the development of IFN-gamma-producing T cells.     

Studies using antigen-presenting cells lacking expression of both B7-1 (CD80) and B7-2 (CD86) show distinct requirements for B7 molecules during priming versus restimulation of Th2 but not Th1 cytokine production

The differentiation of CD4+ T cells into a Th1 vs Th2 phenotype profoundly influences the outcome of autoimmune and infectious diseases. B7 costimulation has been shown to affect the production of both Th1 and Th2 cytokines, depending on the system studied. There is, consequently, great interest in manipulating the B7 costimulatory signal for therapeutic purposes. To optimally manipulate this key immunoregulatory pathway, the contribution of B7 costimulation to cytokine production requires further clarification. We have compared the B7 requirement for cytokine production by naive vs previously activated T cells using DO11.10 TCR transgenic CD4+ T cells and splenic APCs from mice lacking B7 expression. Our data indicate that induction of IL-4 production and Th2 differentiation by naive T cells is highly dependent on B7 molecules, whereas IL-4 production by previously activated T cells is B7 independent. The predominant contribution of B7-mediated signals to Th1 cytokine production by both naive and primed T cells is upon IL-2 production (and expansion) rather than IFN-gamma (effector cytokine) production. Thus, our studies demonstrate that the antigenic experience of a T cell at the time of B7 blockade may determine whether blockade predominantly affects T cell expansion, differentiation, or effector cytokine production. These differential effects of B7 costimulation on IL-2 vs IFN-gamma production and on IL-4 production by naive vs primed T cells have important implications for understanding how B7:CD28/CTLA4 blockade can be effectively used to manipulate cytokine production in vivo.     

Low expression of CD40 and B7 on macrophages infiltrating UV-exposed human skin; role in IL-2Ralpha-T cell activation

After UV exposure of skin, epidermal Langerhans cells (LC) are depleted, whereas CD11b+CD36 CD1a- monocytes/macrophages (UV-Mphi) infiltrate. Different immunological outcomes in vivo are mediated by LC (sensitization) and UV-Mphi (tolerance) which may be related to the distinct T cell activation states that these antigen-presenting cells (APC) induce. We previously demonstrated that CD4+ T lymphocytes activated by UV-Mphi are, in contrast to LC-activated T cells, IL-2Ralpha deficient, and we hypothesize that this differential T cell activation is related to differences in co-stimulatory molecules between UV-Mphi and LC. Using four-color flow cytometry, we found a reduced capacity to up-regulate expression of the important co-stimulatory molecules CD40, B7-1 and B7-2 by UV-Mphi relative to LC. This alteration in co-stimulatory molecule expression was selective, because UV-Mphi express equal levels of ICAM-1 and ICAM-3, and increased levels of LFA-1, relative to LC. After bidirectional signaling with T cells during alloantigen presentation, UV-Mphi still exhibited less CD40 and B7-1 than LC. Addition of IFN-gamma induced CD40 and B7-1 expression on UV-Mphi and restored IL-2Ralpha expression on UV-Mphi-activated T cells but had no effect on IL-2Ralpha on resting or LC-activated T cells. The restoration of IL-2Ralpha expression on UV-Mphi-activated T cells by IFN-gamma was inhibited (67 %, p = 0.005) by addition of neutralizing anti-CD40. Therefore, differences in co-stimulatory molecule expression, in particular CD40, on UV-Mphi and LC are critical in determining the distinct T cell activation induced by these APC.     

Differential interleukin 12 responsiveness for interferon gamma production in advanced stages of cancer patients correlates with performance status

Interleukin 12 (IL-12) has been shown to exhibit potent antitumor activity in murine tumor models through various mechanisms including the capacity to stimulate IFN-gamma production by T cells and natural killer cells. The aim of the present study was to examine the efficacy of IL-12 in inducing IFN-gamma secretion in cancer patients. A comparison was made between healthy individuals who served as controls and cancer patients for IFN-gamma production induced after the stimulation of whole blood samples with 1000 pg/ml IL-12. Samples from all healthy individuals showed positive IL-12 responsiveness. Approximately half of the samples from patients displayed levels of IFN-gamma production comparable to those observed for controls, whereas the rest of the samples exhibited almost-null responses. The incidences for reduced capacity of IFN-gamma production and null IL-12 responsiveness in cancer patients at all cancer stages or at a given advanced stage (stage IV) increased along with performance status. However, these correlated with neither the number of lymphocytes contained in the blood samples nor the tumor types. When peripheral blood mononuclear cells were isolated from patient blood samples showing null/marginal responses, and their responsiveness was examined, 7 of 13 samples exhibited positive responses. Whereas enhanced tumor necrosis factor alpha production was also observed in some patients after IL-12 stimulation, the elevation of tumor necrosis factor alpha was induced only in blood samples that showed IL-12-stimulated IFN-gamma production. These observations indicate that a remarkable difference exists in IL-12 reactivity among cancer patients, and that differential IL-12 responsiveness depends largely on performance status.     

Increased state of activation of CD4 positive T cells and elevated interferon gamma production in pouchitis

We have compared the therapeutic efficacy as well as the kinetics of treatment-induced apoptosis and necrosis of the maximum tolerated dose (MTD) of doxorubicin (DOX) or cisplatin (CDDP) combined with long-duration, low-temperature whole-body hyperthermia (LL-WBH, at 40.0 degrees C for 6 hr), with the combination of the MTDs of either DOX or CDDP with short-duration, high-temperature WBH (SH-WBH, at 41.5 degrees C for 2 hr), in a rat mammary adenocarcinoma (MTLn3). The MTD of LL-WBH + DOX resulted in increased therapeutic efficacy, compared with the MTD of DOX alone and SH-WBH + DOX. The MTD of LL-WBH + CDDP, however, did not increase therapeutic efficacy, when compared with the MTD of CDDP alone or SH-WBH + CDDP. The MTD of LL-WBH + DOX caused a significant delay in the development of spontaneous axillary lymph node (ALN) metastasis and tended to cause longer mean survival, compared with SH-WBH + DOX. The peak of treatment-induced apoptosis was higher for the MTD of DOX + LL-WBH, compared with SH-WBH + DOX, whereas the apoptosis peak of the MTD of SH-WBH + CDDP was higher than that of LL-WBH + CDDP. The most extensive levels of tumor necrosis appeared to occur earlier with SH-WBH alone and the MTD of SH-WBH + DOX or CDDP than with other groups. Our results suggest that LL-WBH + DOX may be a promising therapy for breast cancer, and the extent of treatment-induced tumor apoptosis appears to correlate with antitumor response for MTDs of LL-WBH + DOX and SH-WBH + DOX, but not for the MTDs of CDDP with SH-WBH or LL-WBH.     

Constitutive chemokine production results in activation of leukocyte function-associated antigen-1 on adult T-cell leukemia cells

Adult T-cell leukemia (ATL) is characterized by massive infiltration of circulating ATL cells into a variety of tissues, a finding often associated with poor prognosis. Leukocyte migration from circulation into tissue depends on integrin-mediated adhesion to endothelium, and integrins are tightly regulated by several stimuli, such as inflammatory chemokines. However, the exact mechanisms that enhance adherence of leukemic cells to the endothelium and infiltration into tissues remain to be fully understood. We investigated the mechanisms of extravasation of leukemic cells using ATL cells and report the following novel features of endogenous chemokine-induced adhesion of ATL cells to the endothelium. ATL cells spontaneously adhered to endothelial cells without exogenous stimulation. Integrin leukocyte function-associated antigen-1 (LFA-1) on ATL cells was spontaneously activated. ATL cells produced high amounts of chemokines, macrophage inflammatory protein-1alpha (MIP-1alpha), and MIP-1beta. Adhesion of ATL cells to endothelial cells and the expression of activated form of LFA-1 were reduced by pretreatment with pertussis toxin, wortmannin, or anti-MIP-1alpha and MIP-1beta antibodies or transfection with antisense of MIP-1alpha or MIP-1beta. Spontaneous polymerization of cytoskeletal F-actin was observed in ATL cells, which was also inhibited by pertussis toxin and wortmannin. We propose that ATL cells adhere to endothelial cells through an adhesion cascade similar to normal leukocytes and that the chemokines produced by ATL cells are involved in triggering integrin LFA-1 through cytoskeletal rearrangement induced by G-protein-dependent activation of phosphoinositide 3-kinases in an autocrine manner. These events result in a strong adhesion of ATL cells to the endothelium and spontaneous transendothelial migration.     

Pertussis toxin potentiates Th1 and Th2 responses to co-injected antigen: adjuvant action is associated with enhanced regulatory cytokine production and expression of the co-stimulatory molecules B7-1, B7-2 and CD28

Pertussis toxin (PT) is a major virulence factor of Bordetella pertussis which exerts a range of effects on the immune system, including the enhancement of IgE, IgA and IgG production, delayed-type hypersensitivity reactions, and the induction of experimental autoimmune diseases. However, the mechanism by which PT mediates adjuvanticity remains to be defined. In this investigation we have shown that PT can potentiate antigen-specific T cell proliferation and the secretion of IFN-gamma, IL-2, IL-4 and IL-5 when injected with foreign antigens. A chemically detoxified PT and a genetic mutant with substitutions/deletions in the S-1 and B oligomer components that abrogate enzymatic and binding activity displayed no adjuvant properties. In contrast, a non-toxic S-1 mutant devoid of enzymatic activity but still capable of receptor binding retained its adjuvanticity, augmenting the activation of both Th1 and Th2 subpopulations of T cells. In an attempt to address the mechanism of T cell activation, we found that PT stimulated the production of IFN-gamma and IL-2 by naive T cells and IL-1 by macrophages. Therefore potentiation of distinct T cell subpopulations may have resulted in part from the positive influence of IFN-gamma on the development of Th1 cells and the co-stimulatory role of IL-1 for Th2 cells. Furthermore, PT augmented expression of the co-stimulatory molecules B7-1 and B7-2 on macrophages and B cells, and CD28 on T cells, suggesting that the adjuvant effect may also be associated with facilitation of the second signal required for maximal T cell activation. This study demonstrates that the immunopotentiating properties of PT are largely independent of ADP-ribosyltransferase activity, but are dependent on receptor binding activity and appear to involve enhanced activation of T cells.     

Up-regulation of human epidermal Langerhans' cell B7-1 and B7-2 co-stimulatory molecules in vivo by solar-simulating irradiation

Ultraviolet (UV) radiation impairs cutaneous immune functions and induces antigen-specific tolerance both locally at the irradiated skin site, as well as at distant skin sites and systemically. It has been postulated that in the local model, altered Langerhans' cells (LC) provide tolerogenic signals, and studies in vitro have indicated that UV radiation may down-regulate the expression of co-stimulatory molecules on the surface of these cells. To examine the effect of UV radiation on LC co-stimulatory molecules in vivo, we irradiated human volunteers with erythematogenic doses of solar-simulating UV radiation (SSR), and analyzed the expression of cell surface markers in dermatome skin samples obtained 1-72 h post-irradiation. For flow cytometric analysis, epidermal cell (EC) suspensions were prepared and double labeled with monoclonal antibodies against CD1a or HLA-DR, and B7-1 (CD80), B7-2 (CD86), ICAM-1 (CD54), ICAM-3 (CD50), LFA-3 (CD58), E-cadherin, or integrin-beta4 (CD104). In unirradiated control skin samples, keratinocytes (KC) expressed high levels of E-cadherin. LC expressed high levels of both E-cadherin and ICAM-3, and low levels of B7-2, LFA-3, ICAM-1, and integrin-beta4. Following SSR, a triphasic reaction pattern was seen: an immediate, down-regulatory phase prevailing 2-6 h post-irradiation, when the number of DR+ and CD1a+ cells were temporarily reduced; a delayed, up-regulatory phase in which the number of LC was increased and the expression intensities of CD1a, HLA-DR, B7-1, and B7-2 were strongly up-regulated, maximally evident 12-24 h after irradiation, but no more seen at 48 h; and a late phase at 72 h, in which an influx of monocytes and a concomitant rise in DR+ cells was recorded. We conclude that to understand real-life cutaneous UV immunology, studies in vitro need to be complemented with studies in vivo. In the case of LC, the effects of erythematogenic UV radiation in vivo on human LC B7 co-stimulatory molecules include an up-regulatory stage.     

Generation of nitric oxide and induction of major histocompatibility complex class II antigen in macrophages from mice lacking the interferon gamma receptor

Availability of mice with a targeted disruption of the interferon gamma (IFN-gamma) receptor gene (IFN-gamma R0/0 mice) made it possible to examine parameters of macrophage activation in the absence of a functional IFN-gamma receptor. We asked to what extent other cytokines could replace IFN-gamma in the induction of nitric oxide or major histocompatibility complex class II antigen (Ia) expression in peritoneal macrophages. In thioglycollate-elicited macrophages from wild-type mice, tumor necrosis factor (TNF) alone was virtually ineffective in inducing release of NO2- (the endproduct of nitric oxide generation), but TNF enhanced NO2- release in the presence of IFN-gamma. In macrophages from IFN-gamma R0/0 mice, which were unresponsive to IFN-gamma, TNF completely failed to stimulate NO2- release. The stimulatory actions of IFN-alpha/beta on NO2- release were indistinguishable in wild-type and IFN-gamma R0/0 macrophages: IFN-alpha/beta was ineffective on its own, showed marginal stimulation of NO2- release in combination with TNF, and was moderately effective in the presence of lipopolysaccharide. The level of constitutive Ia antigen expression was not significantly different in peritoneal macrophages from wild-type and IFN-gamma R0/0 mice. An increased Ia expression was induced by IL-4 and granulocyte-macrophage colony-stimulating factor in both wild-type and IFN-gamma R0/0 macrophages, but the magnitude of this induction was less than with optimal concentrations of IFN-gamma in macrophages from wild-type mice. IFN-alpha/beta showed only a minor stimulatory effect on Ia expression in both wild-type and IFN-gamma R0/0 macrophages. Simultaneous treatment of wild-type macrophages with IFN-alpha/beta and IFN-gamma reduced the IFN-gamma-induced Ia expression in wild-type macrophages, but IFN-alpha/beta did not show an inhibitory effect on IL-4- or granulocyte-macrophage-colony-stimulating factor-induced Ia expression in either wild-type or IFN-gamma R0/0 macrophages. The important role of IFN-gamma in the regulation of the induced expression of major histocompatibility complex class II antigen was confirmed by showing that after systemic infection with the BCG strain of Mycobacterium bovis resident peritoneal macrophages from IFN-gamma R0/0 mice had a lower level of Ia expression than macrophages from wild-type mice. The inability of other cytokines to substitute fully for IFN-gamma in macrophage activation helps to explain the earlier observed decreased resistance of IFN-gamma R0/0 mice to some infections.     

Reduced interferon gamma production and soluble CD14 levels in early life predict recurrent wheezing by 1 year of age

It is unknown whether reduced production of IFNgamma in early life, before any lower respiratory tract illness, is a risk factor for recurrent wheezing in infancy. We followed 238 infants prospectively from birth to 1 year of age. At birth and at 3 months of age, IFNgamma production from polyclonally stimulated peripheral blood mononuclear cells and soluble CD14 (sCD14) levels in plasma were measured. The odds of developing recurrent wheezing (assessed by questionnaire) in the first year of life were up to 4.5 times higher for children in the lowest quartile of IFNgamma production at 3 months (p = 0.0005) and 3.2 times higher for children in the lowest quartile of sCD14 levels at birth (p = 0.004) as compared with children in the other 3 combined quartiles of IFNgamma and sCD14, respectively. Findings were confirmed in the multivariate analysis. IFNgamma production at 3 months and sCD14 levels at birth were correlated (r = 0.188, p = 0.031). Our findings from a longitudinal cohort suggest that impaired IFNgamma production at 3 months and reduced plasma-sCD14 levels at birth significantly increase the risk of developing recurrent wheezing in the first year of life.     

p38 MAPK is required for CD40-induced gene expression and proliferation in B lymphocytes

We have investigated the activation of the p38 MAPK pathway in response to CD40 engagement in multiple B cell lines and in human tonsillar B cells to define the role of p38 MAPK in proliferation, NF-kappaB activation and gene expression. Cross-linking CD40 rapidly stimulates both p38 MAPK and its downstream effector, MAPKAPK-2. Inhibition of p38 MAPK activity in vivo with the specific cell-permeable inhibitor, SB203580, under conditions that completely prevented MAPKAPK-2 activation, strongly perturbed CD40-induced tonsillar B cell proliferation while potentiating the B cell receptor (BCR)-driven proliferative response. SB203580 also significantly reduced expression of a reporter gene driven by a minimal promoter containing four NF-kappaB elements, indicating a requirement for the p38 MAPK pathway in CD40-induced NF-kappaB activation. However, CD40-mediated NF-kappaB binding was not affected by SB203580, suggesting that NF-kappaB may not be a direct target for the CD40-induced p38 MAPK pathway. In addition, SB203580 selectively reduced CD40-induced CD54/ICAM-1 expression, whereas CD40-dependent expression of CD40 and CD95/Fas and four newly defined CD40-responsive genes cIAP2, TRAF1, TRAF4/CART and DR3 were unaffected. Our observations show that the p38 MAPK pathway is required for CD40-induced proliferation and that CD40 induces gene expression via both p38 MAPK-dependent and -independent pathways.     

Interleukin 12 acts directly on CD4+ T cells to enhance priming for interferon gamma production and diminishes interleukin 4 inhibition of such priming

Naive CD4+ T cells produce interleukin 2 (IL-2) but little IL-4 or interferon gamma (IFN-gamma). In vitro, they develop into IL-4 or IFN-gamma producers depending on the conditions of the priming culture. Using T-cell receptor transgenic CD4+ T cells, the role of IL-12 and IL-4 in antigen-specific priming was examined. IL-12 substantially enhanced the ability of naive CD4+ T cells to develop into cells that produced IFN-gamma upon restimulation. However, it was not essential since anti-IL-12 antibodies failed to block the priming for IFN-gamma observed in the absence of exogenous IL-12. When both IL-12 and IL-4 were present in the priming culture, IL-12 did not inhibit priming for IL-4 production. In contrast, IL-4 diminished but did not abolish priming for IFN-gamma production. In an accessory cell-independent priming system, IL-12 strikingly augmented priming for IFN-gamma production, indicating that it acts directly on T cells. IFN-gamma itself did not enhance priming for IFN-gamma production in either accessory cell-dependent or independent systems. In an accessory cell-dependent system, the IL-12-mediated enhancement was not blocked by adding neutralizing anti-IFN-gamma monoclonal antibody. However, in an accessory cell-independent system, anti-IFN-gamma antibody did inhibit priming for IFN-gamma production leaving open a role for IFN-gamma in the priming process. These data indicate that IL-12 has a major effect on the inductive phase of T-cell priming by enhancing commitment to IFN-gamma production and thus can profoundly influence the state of immunity that develops.     

Signal transduction by CD28 costimulatory receptor on T cells. B7-1 and B7-2 regulation of tyrosine kinase adaptor molecules

This study compares the biochemical responses in T cells activated with the CD28 ligands B7-1 and B7-2. The patterns of tyrosine phosphorylation induced in T cells by these two CD28 ligands are identical, but clearly different from the tyrosine phosphorylation induced by the T cell receptor (TCR). The TCR regulates protein complexes mediated by the adapter Grb2 both in vivo and in vitro. In contrast, there is no apparent regulation of in vivo Grb2 complexes in response to B7-1 or B7-2. Rather, B7-1 and B7-2 both induce tyrosine phosphorylation of a different adaptor protein, p62. The regulation of p62 is a unique CD28 response that is not shared with the TCR. These data indicate that B7-1 and B7-2 induce identical tyrosine kinase signal transduction pathways. The data show also that the TCR and CD28 couple to different adapter proteins, which could explain the divergence of TCR and CD28 signal transduction pathways during T cell activation.     

Effects of liposome-encapsulated hemoglobin on phorbol ester-induced superoxide production and expression of costimulatory molecules by monocytes in vitro

The effects of Neo Red Cell (NRC), a liposome-encapsulated hemoglobin (LEH), on the phorbol ester-induced superoxide production and the expression of costimulatory molecules by human peripheral monocytes were investigated. The treatment of human mononuclear cells with NRC caused the potentiation of superoxide production in response to PMA. The longer incubation (20 h) resulted in a decrease in the PMA-induced superoxide production, which was in parallel to a decrease in the viability of the monocytes. A flow cytometric analysis showed that a slight expression of CD80 (B7-1) on monocytes was induced by NRC treatment, whereas the constitutive expressions of CD86 (B7-2) and CD54 (ICAM-1) were unchanged. The activation of monocytes with interferon-gamma (IFN-gamma) induced the expressions of CD80, CD86, and CD54 under all conditions tested, but NRC treatment tended to decrease the IFN-gamma-induced expression of CD54 on monocytes. These results suggest that the administration of LEH may modify the functions of human monocytes.     

Tumor necrosis factor soluble receptor 75: the principal receptor form released by human alveolar macrophages and monocytes in the presence of interferon gamma

Tumor necrosis factor alpha(TNF alpha), a proinflammatory cytokine secreted predominantly by monocytemacrophages, interacts with two cell-surface receptors: TNF-R55 and TNF-R75. Few studies have been devoted to their modulation on human alveolar macrophages (AM). Both source and target of TNF(alpha), AM also release its inhibitors, the soluble receptors, following the cleavage of the extracellular domain of TNF-R55 and TNF-R75. Because in vivo AM are subject to activation by exogenous or endogenous stimuli, we analyzed the release of both receptors into the cell culture supernatant in response to lipopolysaccharide (LPS), phorbol myristate acetate (PMA), and cytokines such as interleukin 2(IL-2), IL-4, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interferon gamma (IFN-gamma). Results were compared with those obtained on peripheral blood monocytes (Mo), and the role of receptor recycling was investigated using inhibitors such as monensin and chloroquine. In our culture conditions, basal release by unstimulated AM amounted to 0.3 +/- 0.1 and 0.5 +/- 0.1 ng/ml for TNF-sR55 and TNF-sR75, respectively. In the same conditions, Mo released 1.2 +/- 1.2 ng/ml of TNF-sR55 and 5.1 +/- 0.1 ng/ml of TNF-sR75. PMA slightly increased mRNA expression and release of TNF-sR55, but those of TNF-sR75 were enhanced approximately 4-fold. After 24 h of culture, the release of TNF-sR75 was 2.5-fold higher on Mo than on AM. Of the cytokines tested on AM, IFN-gamma increased the release of TNF-sR75 3-fold, but that of TNF-sR55 only between 1.5- and 2-fold. GM-CSF enhanced them to a lower extent (approximately 1.5-fold). Shedding occurred despite the presence of chloroquine, monensin and colchicine, suggesting that cleavage takes place on the cell surface rather than after internalization. Addition of colchicine increased the release of TNF-sR75 induced by LPS and IFN-gamma, but not by PMA. In conclusion, Mo and AM differ in their ability to release TNF(alpha) and TNF-sR. On AM the release of each receptor appears to be regulated separately. Finally, IFN-gamma was among the most efficacious cytokines to induce the release of both receptors, with TNF-sR75 being more liable to shedding. Thus, the two TNF-R seem to be ruled by separate mechanisms and to differ in terms of release sensitivity.