Human B cell responsiveness to B cell growth factor after activation by phorbol ester and monoclonal anti-mu antibody

The effect of phorbol ester on human B cell activation was examined. Picomolar to nanomolar concentrations of phorbol ester induced a high level of proliferation in small IgM-positive B cells isolated from peripheral blood by fluorescence-activated cell sorting. The addition of optimal doses of anti-mu antibody resulted in enhanced proliferation of phorbol ester-activated B cells. The addition of B cell growth factor (BCGF) to phorbol ester-activated B cells also resulted in a dose-dependent synergistic effect and maximal enhancement on day 3. BCGF activity could be absorbed with either phorbol ester- or anti-mu-activated B cells, but not with resting B cells, thus confirming the induction of functional BCGF receptor expression. Cell proliferation was not necessary for the induction of functional BCGF receptors. Phorbol ester was a more efficient inducer of BCGF receptor expression than was anti-mu antibody; gamma-interferon treatment had no effect. BCGF enhanced transferrin receptor expression by phorbol ester-activated B cells. The results suggest that phorbol ester-activated small B cells can be used to monitor BCGF activity, and this synergistic combination may be useful in establishing BCGF-dependent B cell clones in culture.     

Mycobacterium avium infection of mouse macrophages inhibits IFN-gamma Janus kinase-STAT signaling and gene induction by down-regulation of the IFN-gamma receptor.

Macrophage activation is required to control the growth of intracellular pathogens. Recent data indicate that macrophages become functionally deactivated during mycobacterial infection. We studied macrophage deactivation by examining the expression of a panel of IFN-gamma-inducible genes and activation of Janus Kinase (JAK)-STAT pathway in Mycobacterium avium-infected macrophages. Reduced expression of IFN-gamma-inducible genes-MHC class II gene E beta; MHC class II transactivator; IFN regulatory factor-1; and Mg21, a gene coding for a GTP-binding protein-was observed in M. avium-infected macrophages. Decreased tyrosine phosphorylation and DNA binding activity of STAT1 in M. avium-infected macrophages stimulated with IFN-gamma was observed. Tyrosine phosphorylation of JAK1, JAK2, and IFN-gamma R alpha was also reduced in infected cells. Northern and Western blot analyses showed that a down-regulation of IFN-gamma R alpha- and beta-chain mRNA and protein occurred in M. avium-infected macrophages. The down-regulation of IFN-gamma R and inhibition of STAT1 activation were time dependent and required 4 h of infection for down-regulation of the IFN-gamma R and 8 h for STAT1 inhibition. These findings suggest that M. avium infection inhibits induction of IFN-gamma-inducible genes in mouse macrophages by down-regulating IFN-gamma R, resulting in reduced phosphorylation of IFN-gamma R alpha, JAK1, JAK2, and STAT1.     

High level class II trans-activator induction does not occur with transient activation of the IFN-gamma signaling pathway

Gene activation in early development is highly dependent on precise concentrations of trans-acting factors for the activation of different genes at differing points in the embryo. Thus, not only is the presence or absence of a particular trans-activator or repressor relevant in determining gene activation, but also the concentration of the regulatory protein must be above or below a certain threshold for proper gene regulation. Signaling pathways in somatic cells are thought to represent cascades of on/off switches, mediated most commonly by phosphorylation. Here we demonstrate a quantitative mechanism for regulating the level of a component of the IFN-gamma signaling pathway that in effect represents the differential sensitivities of STAT1, IFN-regulatory factor-1, and class II trans-activator (CIITA) to IFN-gamma. Unlike developmental gene regulation, in which specificity of gene activation is a function of regulatory protein concentrations, specificity of gene activation in the IFN-gamma signaling pathway is regulated by the duration of the activation of the primary IFN-gamma-regulatory protein, STAT1. This result most likely explains previously reported data indicating that a minimum amount of IFN-gamma is required for MHC class II gene activation despite the fact that the level of the IFN-gamma-inducible factor directly required for MHC class II induction, CIITA, directly correlates with the level of MHC class II expression. The induction of a high level of CIITA is dependent on sustained IFN-gamma signaling. The possible implications of this result for tumorigenesis are discussed.     

Invariant chain influences post-translational processing of HLA-DR molecules

HLA class II MHC molecule alpha- and beta-chains are normally synthesized in the presence of a third molecule, the invariant chain (Ii). Although Ii is not required for surface expression of HLA class II molecules, the influence of Ii on post-translational processing and maturation HLA class II molecules has not been thoroughly studied. In the present study, BALB/c 3T3 cells were transfected with HLA-DR alpha- and beta-chains with or without co-transfection with human Ii. Although Ii had no effect on the surface expression of DR, Ii did have a profound effect on the post-translational processing of both the alpha- and beta-chains. In the absence of Ii, the major species of alpha- and beta-chains were of lower m.w. than when expressed in the presence of Ii. The differences in m.w. were shown to be caused by differences in glycosylation with the majority of alpha- and beta-chains remaining unprocessed and endo H sensitive in the absence of Ii. The small proportion of alpha-chains that were processed in the absence of Ii showed an altered m.w. and altered sensitivity to treatment with endo H relative to alpha-chains processed in the presence of Ii. Pulse/chase studies demonstrated that although the majority of the alpha- and beta-chains remained unprocessed in the absence of Ii, the small amount that was processed was done so at a rate similar to that observed for alpha- and beta-chains processed in the presence of Ii. These studies demonstrate that Ii influences the post-translational processing of human class II molecules by affecting the proportion of alpha- and beta-chains that are processed and by determining the degree of processing of oligosaccharides on mature alpha-chains.     

Regulation of MHC class II antigen expression. Opposing effects of tumor necrosis factor-alpha on IFN-gamma-induced HLA-DR and Ia expression depends on the maturation and differentiation stage of the cell

MHC class II induction by cytokines has been suggested to play a major role in the initiation and propagation of immune and autoimmune processes. TNF-alpha has been found both to enhance and also to inhibit IFN-gamma-induced MHC class II expression. In the present studies, the effect of TNF-alpha on IFN-gamma induced MHC class II expression was tested in various cell lines. On the basis of the data, we propose that, depending on the stage of differentiation and maturation of the cells, TNF-alpha might synergize or antagonize the affects of IFN-gamma on the regulation of MHC class II expression. Thus, in immature cells such as HL-60 or THP-1, TNF-alpha enhances IFN-gamma-induced class II expression. However, when differentiation was induced in these cells by TPA or IFN-gamma, the additive effect of TNF-alpha on the IFN-gamma induced DR expression was eliminated. Furthermore, TNF-alpha down-regulates the IFN-gamma-induced class II expression in differentiated cells such as human skin fibroblasts or activated macrophages. In bone marrow cells induced to differentiate in vitro, TNF-alpha decreased the IFN-gamma-induced MHC class II expression in a maturation-dependent fashion. These results provide a rational explanation for the conflicting reports regarding the effect of TNF-alpha on IFN-gamma-induced class II expression. But more importantly they may be relevant to the biologic function of TNF-alpha. Thus, we show that TNF-alpha-treated mice have reduced level of Ia expression on peritoneal macrophages and in vivo treatment with TNF-alpha antagonizes the ability of IFN-gamma to induce class II expression on these macrophages.     

Cathepsin B cleavage of Ii from class II MHC alpha- and beta-chains

Class II MHC-associated invariant chain (Ii) might regulate binding of digested peptides to the Ag binding site (desetope) of class II MHC proteins by directly or allosterically blocking that site until cleavage and release of Ii from MHC alpha- and beta-chains at the time of peptide charging. We examined the cleavage and release of Ii from class II MHC alpha/beta Ii trimers by cathepsin B, which has been shown by others to colocalize with class II MHC molecules in intracellular compartments and to generate antigenic peptide fragments. Cathepsin B at pH 5.0 cleaved and released Ii from class II MHC alpha- and beta-chains. Cathepsin B digested Ii from alpha- and beta-chains in a dose-dependent fashion, yielding 23-, 21-, and 10-kDa fragments. Blockage of cathepsin B activity with leupeptin restored the 2D(nonequilibrium pH gradient gel electrophoresis/SDS) PAGE patterns of Ii and sialic acid-derivatized forms of Ii seen without the protease. The fragmentation pattern of cathepsin D treatment was different from that of cathepsin B, yielding 25-kDa intermediates.     

A positive regulatory role for suppressor of cytokine signaling 1 in IFN-gamma-induced MHC class II expression in fibroblasts

Suppressor of cytokine signaling 1 (SOCS1) is rapidly induced following stimulation by several cytokines. SOCS1 negatively regulates cytokine receptor signal transduction by inhibiting Janus family tyrosine kinases. Lack of such feedback regulation underlies the premature death of SOCS1(-/-) mice due to unbridled IFN-gamma signaling. We used mouse embryo fibroblasts derived from SOCS1(-/-) mice to investigate the role of SOCS1 in IFN-gamma signaling pathways. SOCS1(-/-) fibroblasts were exquisitely sensitive to the IFN-gamma-mediated growth arrest and showed sustained STAT1 phosphorylation. However, SOCS1(-/-) fibroblasts were inefficient in MHC class II surface expression following IFN-gamma stimulation, despite a marked induction of the MHC class II transactivator and MHC class II gene expression. Retroviral transduction of wild-type SOCS1 relieved the growth-inhibitory effects of IFN-gamma in SOCS1(-/-) fibroblasts by inhibiting STAT1 activation. SOCS1R105K, carrying a mutation within the phosphotyrosine-binding pocket of the Src homology 2 domain, did not inhibit STAT1 phosphorylation, yet considerably inhibited IFN-gamma-mediated growth arrest. Strikingly, expression of SOCS1R105K restored the IFN-gamma-induced MHC class II expression in SOCS1(-/-) cells, indicating that expression of SOCS1 facilitates MHC class II expression in fibroblasts. Our results show that SOCS1, in addition to its negative regulatory role of inhibiting Janus kinases, has an unanticipated positive regulatory function in retarding the degradation of IFN-gamma-induced MHC class II proteins in fibroblasts.     

Granulocyte-macrophage colony stimulating factor induces both HLA-DR expression and cytokine production by human monocytes

The effect of recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) on the expression of HLA-DR, and the production of the cytokines interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha) by human peripheral blood monocyte-enriched populations was investigated. GM-CSF was shown to induce both the expression of HLA-DR and the cytokines IL-1 and TNF alpha in a dose-dependent manner. In contrast, interferon-gamma (IFN-gamma), which induced major histocompatibility complex (MHC) class II expression, did not induce IL-1 or TNF alpha production. However, IFN-gamma enhanced the cell surface expression of HLA-DR and the production of IL-1 and TNF alpha on monocyte-enriched cells stimulated by GM-CSF. By itself, GM-CSF did not induce surface class II expression on the human monocytic tumour cell line THP-1, whereas it synergized with IFN-gamma to induce surface expression. These cells responded to GM-CSF by producing IL-1 and TNF alpha; Northern blotting showed that mRNA levels of IL-1 and TNF alpha were transiently induced, similar to other cytokines. Our results indicate that GM-CSF is a major macrophage activating factor that is capable of inducing both the expression of HLA-DR and the cytokines involved in T-cell activation by macrophages; therefore, GM-CSF may be of importance in potentiating antigen presenting function.     

17beta-estradiol inhibits NADPH oxidase activity through the regulation of p47phox mRNA and protein expression in THP-1 cells

In this report, we demonstrate that NADPH oxidase is activated by tumor necrosis factor-alpha (TNF-alpha) plus interferon-gamma (IFN-gamma) in human monocytic cells (THP-1 cells) differentiated with phorbol ester (PMA) and that physiological concentration of 17beta-estradiol inhibits NADPH oxidase activity in THP-1 cells stimulated with TNF-alpha plus IFN-gamma. This effect is mediated by estrogen receptor based on estrogen receptor antagonist (ICI 182, 780) that diminishes inhibition by 17beta-estradiol. This inhibition is specific in 17beta-estradiol because 17alpha-estradiol, testosterone and progesterone do not inhibit NADPH oxidase activity. Activation of NADPH oxidase induced by TNF-alpha plus IFN-gamma is caused by up-regulation of p47(phox) (cytosolic component of NADPH oxidase) expression. 17beta-Estradiol prevents the up-regulation of p47(phox) mRNA and protein expression. This prevention of p47(phox) expression depends on the inhibition of NF-kappaB activation. Our results implicate that 17beta-estradiol has an anti-atherosclerotic effects through the improvement of nitric oxide (NO) bioavailability caused by the regulation of superoxide (O(2)(-)) production.     

Rabbit MHC. II. Sequence analysis of the R-DP alpha- and beta-genes

Molecular genetics studies have recently revealed complexity in the MHC class II region that has not been detected by previous serologic and genetic studies. In humans, three subregions, DP, DQ, and DR, of the class II genes as well as the DZ alpha and DO beta genes, have been extensively characterized. Although homologs of these human genes were identified in many species, their expressibility has not been well defined in species other than the mouse. We have previously cloned the rabbit homologs of the HLA-DP alpha and beta genes whose protein products had never been detected. The sequences of rabbit DP alpha 1 and DP beta genes are reported herein and they indicate that the rabbit DP genes encode functional alpha- and beta-chains. Unfavorable nucleotides surrounding the first AUG codon may, however, reduce the translational efficiency of the R-DP beta mRNA and explain the difficulty in generating serologic reagents specific for rabbit DP molecules. A complex mutation in the beta 1 domain of the R-DP beta gene was similar to the one found in the H-2A beta 1 gene of five strains of mice. The origin of this mutation is discussed.