Human peripheral blood monocytes release a 30,000 dalton factor (30 KD MF) that stimulates immunoglobulin production by activated B cells

Supernatants (SN) of well-washed adherent human monocytes, obtained from T cell-depleted peripheral blood mononuclear cells, contain a 30,000 dalton protein (30 KD MF) that increases immunoglobulin (Ig) synthesis by EBV-activated B cells two- to fourfold. This factor is released spontaneously during the first 20 hr after monocytes are placed in culture. SN containing 30 KD MF are inactive in the thymocyte co-stimulator assay, under conditions that will detect as little as 0.5 U of purified IL 1. The addition of autologous T cells to isolated adherent monocytes, previously depleted of T cells, suppresses the release or activity of this B cell stimulator in a dose-dependent manner. In addition, 30 KD MF stimulates a two- to fourfold increase in IgA production by cells of an EBV-transformed B cell line (JB/FF line) without increasing incorporation of [3H]thymidine. In contrast, stimulation of this B cell line with up to 10 U of purified IL 1 increases IgA synthesis by less than 50%, and addition of up to 100 U of recombinant IL 2 causes no change whatsoever in IgA production. However, co-stimulation with 30 KD MF and recombinant IL 2 or recombinant gamma-interferon induces more Ig production than is caused by the monocyte factor alone. These observations suggest that the monocyte, in addition to acting as an antigen-presenting cell and source of IL 1, facilitates B cell differentiation by producing a factor which acts both independently and in synergy with cytokines produced by T cells to stimulate Ig production by B lymphocytes.     

Cyclic AMP-mediated modulation of immunoglobulin production in B cells by prostaglandin E1.

We had previously demonstrated in a transformed human B cell line, LA350, the existence of an inverse relationship between cyclic adenosine monophosphate (cAMP) content and immunoglobulin secretion using the cAMP-elevating agents such as cholera toxin and forskolin. In this paper we report that cAMP acting as a second messenger for prostaglandin exerts a similar effect on the antibody response of B lymphocytes. Incubation of the cells with PGE1 in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) produced a concentration- and time-dependent elevation of intracellular cAMP. Significant increases of cAMP production were observed at physiologically relevant levels of PGE1 (10(-7) and 10(-8) M). Immunoglobulin production, whether measured as the total number of immunoglobulin-secreting cells by a reverse hemolytic plaque assay or as specific immunoglobulin production (IgM) by an enzyme-linked immunoadsorbent assay, was suppressed in a dose-dependent fashion by the presence of IBMX. This suppression of immunoglobulin production was significantly enhanced by the presence of PGE1. Phorbol myristate acetate-induced IgM production was also inhibited by the presence of PGE1. These results imply that prostaglandins regulate B cell activation and immunoglobulin production by signal transduction mechanisms involving cyclic nucleotides.     

Axons and myelinating glia: An intimate contact

The coordination of the vertebrate nervous system requires high velocity signal transmission between different brain areas. High speed nerve conduction is achieved in the myelinated fibers of both the central and the peripheral nervous system where the myelin sheath acts as an insulator of the axon. The interactions between the glial cell and the adjacent axon, namely axo-glial interactions, segregate the fiber in distinct molecular and functional domains that ensure the rapid propagation of action potentials. These domains are the node of Ranvier, the paranode, the juxtaparanode and the internode and are characterized by multiprotein complexes between voltage-gated ion channels, cell adhesion molecules, members of the Neurexin family and cytoskeletal proteins. In the present review, we outline recent evidence on the key players of axo-glial interactions, depicting their importance in myelinated fiber physiology and disease.     

Purification and characterization of immunoglobulin production stimulating factor derived from human B lymphoblastoid HO-323 cells

An immunoglobulin (Ig) production stimulating factor (IPSF) for hybridomas was found in spent medium of the human B lymphoblastoid cell line, HO-323. The IPSF was purified by serial use of DEAE chromatography, ultrafiltration, gel filtration and HPLC-DEAE chromatography. Purified IPSF was estimated to be a 410 k macro molecule by gel filtration, and contained three types of isomers which were separated from each other by native polyacrylamide gel electrophoresis. All of the isomers were, however, assumed to have the same protein components by SDS polyacrylamide gel electrophoresis.The IPSF was effective for human-human and mouse-mouse hybridomas producing IgM, but not for IgG producers in the experimental condition used here. Human-human hybridoma HF10B4, cultured in IPSF-containing medium, produced 20 times more IgM than in IPSF-free medium under serum-free conditions. The IPSF showed very little proliferation stimulating activity on HF10B4 cells.     

Clustering of neuronal sodium channels requires contact with myelinating Schwann cells

Efficient and rapid conduction of action potentials by saltatory conduction requires the clustering of voltage-gated sodium channels at nodes of Ranvier. This clustering results from interactions between neurons and myelinating glia, although it has not been established whether this glial signal is contact-dependent or soluble. To investigate the nature of this signal, we examined sodium channel clustering in co-cultures of embryonic rat dorsal root ganglion neurons and Schwann cells. Cultures maintained under conditions promoting or preventing myelination were immunostained with antibodies against the α subunit of the sodium channel and against ankyrin_G, a cytoskeletal protein associated with these channels. Consistent with previous in vivo studies (Vabnick et al., 1996), sodium channels and ankyrin G cluster at the onset of myelination. These clusters form adjacent to the ends of the myelinating Schwann cells and appear to fuse to form mature nodes. In contrast, sodium channels and ankyrin G do not cluster in neurons grown alone or in co-cultures where myelination is precluded by growing cells in defined media. Conditioned media from myelinating co-cultures also failed to induce sodium channel or ankyrin G clusters in cultures of neurons alone. Finally, no clusters develop in the amyelinated portions of suspended fascicles of dorsal root ganglia explants despite being in close proximity to myelinated segments in other areas of the dish. These results indicate that clustering of sodium channels requires contact with myelinating Schwann cells.     

Activation of immunoglobulin allotype-specific T cells by B cells

To investigate the role of Ia and immunoglobulin (Ig) molecules of B cells in alloantigen-specific and nominal antigen-specific T-cell activations, the ability of B cells to stimulate Ig allotype-specific T cells was examined. T15-primed B10.BR T cells responded to MOPC 315 (IgA myeloma protein derived from BALB/c) as well as T15 but not to MOPC31c (IgG, myeloma protein). These T cells were stimulated by papain-digested Fc fragment of T15. Thus, T15-primed B10.BR T cells were shown to be specific for Ig allotype of T15, that is, Igh-2a. T15-specific B10.BR T cells were selected by 10-day cultures with T15 in vitro. They responded to BALB.K spleen cells without addition of soluble T15 antigen to the assay culture. Stimulator cells in this mixed lymphocyte reaction (MLR)-like response between T15-specific B10.BR T cells and BALB.K spleen cells were Thy-1⁻, Ia⁺ cells and these responses were blocked by anti-Ia^κ antibodies. Furthermore, Sephadex G-10-passed BALB.K B cells stimulated the proliferation of T15-specific B10.BR T cells, while they failed to stimulate allogeneic BALB/c spleen cells. The stimulating ability of B cells in this MLR-like response of T15-specific B10.BR T cells was shown to be genetically restricted, namely, both H-2 and non-H-2 genes are involved in the manifestation of the stimulating ability. This system will provide a useful model for studying the role of B-cell surface Ig and Ia molecules in the activation of antigen-specific T cells and alloreactive T cells.     

Interaction between transmembrane TNF and TNFR1/2 mediates the activation of monocytes by contact with T cells

Monocytes and monocytic cells produce proinflammatory cytokines upon direct cell contact with activated T cells. In the autoimmune disease rheumatoid arthritis, the pivotal role of TNF-alpha implies that the interaction between transmembrane TNF-alpha (mTNF) and the TNF receptors (TNFR1 and TNFR2) might participate in the T cell contact-dependent activation of monocytes. Accordingly, treatment of rheumatoid arthritis by administration of a TNF-alpha-blocking Ab was found to significantly decrease TNF-alpha production by monocytes. Several lines of evidence indicated that signaling through TNFR1/2 and through mTNF (reverse signaling) is involved in TNF-alpha production by monocytes after T cell contact: 1) blocking mTNF on activated T cells leads to a significant reduction in TNF-alpha production; 2) down-regulation of TNFR1/2 on monocytes by transfection with small interfering RNA results in diminished TNF-alpha production; 3) blocking or down-regulating TNFR2 on activated T cells inhibits TNF-alpha production, indicating that mTNF on the monocyte surface mediates signaling; 4) ligation of mTNF on monocytes by surface TNFR2 transfected into resting T cells induces TNF-alpha production due to reverse signaling by mTNF; and 5) ligation of mTNF on monocytes by a soluble TNFR2:Ig receptor construct induces TNF-alpha production due to reverse signaling. In conclusion, we identified mTNF and TNFR1/2 as interaction partners contributing to TNF-alpha production in monocytes. Both pathways initiated by mTNF-TNFR interaction are likely to be inhibited by treatment with anti-TNF-alpha Abs.     

The effect of 1,25-dihydroxyvitamin D3 on in vitro immunoglobulin production in human B cells

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) dose-dependently suppressed immunoglobulin (Ig) production of human B cells, as evaluated by IgG-plaque-forming cells (IgG-PFC) in the culture of pokeweed mitogen (PWM)-activated B cells. Similar suppressive effect of 1,25(OH)2D3 on Ig production of B cells was observed in the Staphylococcus aureus Cowan I(SAC)-induced Ig-producing system. The mean percentage of inhibitions at a concentration of 10(-9) M were 60.0 +/- 8.2% (mean +/- SE, n = 6) and 65.1 +/- 4.7% (n = 10) in PWM- and SAC-stimulated cultures, respectively. The suppression was strongly exhibited only when 1,25(OH)2D3 was added at the start of the 6-day culture, accompanied by a decrease in DNA synthesis of B cells in both culture systems. On the other hand, the addition of 1,25(OH)2D3 on day 4, when DNA synthesis reached at plateau and IgG-PFC began to be detectable, had no noticeable affect on either the number of PFC or DNA synthesis of B cells. Furthermore, 1,25(OH)2D3 suppressed Ig production even when B cells were exposed to the agent for 4 hr after the activation with PWM or SAC, but not before the activation. These results indicate that 1,25(OH)2D3 inhibits B cell proliferation before differentiation to Ig-secreting cells, consequently reducing Ig production; and that its action appears to be mediated by the cytosol receptors expressed on activated B cells. Thus, the agent may serve as an immunoregulating hormone in vivo, as well as in vitro.     

Relationship between immunoglobulin production and immortalization by Epstein Barr virus

After infection with Epstein Barr virus (EBV), human B lymphocytes actively secrete immunoglobulin (Ig) and are immortalized to become long-term cell lines. In these studies, we investigated the relationship between these virally induced processes utilizing limiting dilution culture techniques, and asked whether all B cells stimulated by EBV to secrete Ig are also immortalized. The activation of B cells by EBV resulting in Ig production and immortalization involved a single precursor cell, required live viral particles, and was independent of immunity to EBV by the lymphocyte donor. However, the precursor frequency of B cells activated to secrete Ig (mean 4.7%) was higher than the precursor frequency of B cells activated to long-term in vitro growth (mean 2.1%). When examined at a single cell level, it appeared that although the vast majority of the immortalized B cells also secrete Ig, only approximately 50% of the B cell precursors induced by EBV to secrete Ig go on to form long-term cell lines. In addition, although immortalized B cell clones producing all major classes of Ig were detected, IgM-committed precursors were more likely to become immortal than were precursors committed to IgG or IgA production. In contrast to these findings in B cells freshly infected with EBV, Ig production was almost always associated with evidence of long-term growth when B cells from previously established EBV-induced B cell lines were tested in identical limiting dilution cultures. Thus, after infection with EBV, human B cells can either become transiently activated to proliferate and to secrete Ig, or become transformed into long-term cell lines most of which produce Ig.     

Measles virus spread between neurons requires cell contact but not CD46 expression, syncytium formation, or extracellular virus production

In patients with subacute sclerosing panencephalitis (SSPE), which is associated with persistent measles virus (MV) infection in the brain, little infectious virus can be recovered despite the presence of viral RNA and protein. Based on studies of brain tissue from SSPE patients and our work with MV-infected NSE-CD46(+) mice, which express the measles receptor CD46 on neurons, several lines of evidence suggest that the mechanism of viral spread in the central nervous system differs from that in nonneuronal cells. To examine this alternate mechanism of viral spread, as well as the basis for the loss of normal transmission mechanisms, infection and spread of MV Edmonston was evaluated in primary CD46(+) neurons from transgenic mice and differentiated human NT2 neurons. As expected, unlike that between fibroblasts, viral spread between neurons occurred in the absence of syncytium formation and with minimal extracellular virus. Electron microscopy analysis showed that viral budding did not occur from the neuronal surface, although nucleocapsids were present in the cytoplasm and aligned at the cell membrane. We observed many examples of nucleocapsids present in the neuronal processes and aligned at presynaptic neuronal membranes. Cocultures of CD46(+) and CD46(-) neurons showed that cell contact but not CD46 expression is required for MV spread between neurons. Collectively, these results suggest that the neuronal environment prevents the normal mechanisms of MV spread between neurons at the level of viral assembly but allows an alternate, CD46-independent mechanism of viral transmission, possibly through the synapse.     

Elevated proinflammatory cytokine production by a skewed T cell compartment requires monocytes and promotes inflammation in type 2 diabetes

An appropriate balance between proinflammatory (Th17 and Th1) and anti-inflammatory (regulatory T cells [Tregs] and Th2) subsets of T cells is critical to maintain homeostasis and avoid inflammatory disease. Type 2 diabetes (T2D) is a chronic inflammatory disease promoted by changes in immune cell function. Recent work indicates T cells are important mediators of inflammation in a mouse model of T2D. These studies identified an elevation in the Th17 and Th1 subsets with a decrease in the Treg subset, which culminates in inflammation and insulin resistance. Based on these data, we tested the hypothesis that T cells in T2D patients are skewed toward proinflammatory subsets. Our data show that blood from T2D patients has increased circulating Th17 cells and elevated activation of Th17 signature genes. Importantly, T cells required culture with monocytes to maintain Th17 signatures, and fresh ex vivo T cells from T2D patients appeared to be poised for IL-17 production. T cells from T2D patients also have increased production of IFN-γ, but produce healthy levels of IL-4. In contrast, T2D patients had decreased percentages of CD4(+) Tregs. These data indicate that T cells in T2D patients are naturally skewed toward proinflammatory subsets that likely promote chronic inflammation in T2D through elevated cytokine production. Potential therapies targeted toward resetting this balance need to be approached with caution due to the reciprocal relationship between Th17 cells and Tregs. Understanding the unique aspects of T2D T cells is essential to predict outcomes of such treatments.     

IL-33 activates B1 cells and exacerbates contact sensitivity

B1 B cells produce natural IgM and play a critical role in the early defense against bacterial and viral infection. The polyreactive IgM also contributes to the clearance of apoptotic products and plays an important role in autoimmune pathogenesis. However, the mechanism of activation and proliferation of B1 cells remains obscure. In this study, we report that IL-33, a new member of IL-1 family, activates B1 cells, which express the IL-33 receptor α, ST2. IL-33 markedly activated B1 cell proliferation and enhanced IgM, IL-5, and IL-13 production in vitro and in vivo in a ST2-dependent manner. The IL-33-activated B1 cell functions could be largely abolished by IL-5 neutralization and partially reduced by T cell or mast cell deficiency in vivo. ST2-deficient mice developed less severe oxazolone-induced contact sensitivity (CS) than did wild-type (WT) mice. Furthermore, IL-33 treatment significantly exacerbated CS in WT mice with enhanced B1 cell proliferation and IgM and IL-5 production. Moreover, IL-33-activated B1 cells from WT mice could adoptively transfer enhanced CS in ST2(-/-) mice challenged with IL-33. Thus, we demonstrate, to the best of our knowledge, a hitherto unrecognized mechanism of B1 cell activation and IL-33 function, and suggest that IL-33 may play an important role in delayed-type hypersensitivity.     

Regulation of soluble and surface-bound TRAIL in human T cells, B cells, and monocytes

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF/nerve growth factor superfamily that, apart from inducing cell death in susceptible cells, displays immunoregulatory functions influencing, for instance, T cell proliferation. It can be found in two forms: membrane-bound and soluble protein. The regulation of these is still not fully understood. In this study, we have analyzed the regulation of TRAIL surface expression and secretion in human T cells, B cells, and monocytes in response to specific stimuli. T cells, B cells, and monocytes were cultured in the presence of phytohemagglutinin (PHA)+interleukin (IL-2), anti-CD40+IL-4, and lipopolysaccharide (LPS), respectively. In particular, not only PHA+IL-2 but also LPS were able to induce secretion of soluble TRAIL, but did not enhance the expression of surface-bound TRAIL. Simultaneously, we investigated the effect of the pleiotropic stimulus interferon (IFN)-beta, known to target all leukocyte subsets, on TRAIL. Predominantly, monocytes were affected by IFN-beta, causing both release of soluble TRAIL and upregulation of the surface-bound form. IFN-beta, however, did not cause any upregulation of TRAIL in T cells. Our data serve as a basis to better understand the complex regulation of TRAIL in human peripheral immune cells and might help to clarify the role of the TRAIL system in immunopathology.     

Differential induction of interleukin-6 production in monocytes, endothelial cells and smooth muscle cells.

Studying the production of IL-6 (interleukin-6) by monocytes, endothelial cells and smooth muscle cells we observed that cytokine inducers like IL-1, TNF alpha (tumor necrosis factor alpha), LPS (lipopolysaccharide), SAC (Staphylococcus Aureus Cowan 1) and PMA could be divided roughly into two categories. Bacterial products such as LPS or SAC have a potent IL-6 inducing effect on monocytes and minor or no effect on endothelial- and smooth muscle cells. The other category comprising IL-1, TNF alpha and PMA induces IL-6 production in endothelial- and smooth muscle cells. Only IL-1 induces IL-6 production in monocytes as well as in endothelial cells and smooth muscle cells. In addition to IL-6, also IL-1 and TNF alpha are produced by monocytes however with different kinetics. None of the stimuli had any inhibitory effect on IL-6 production with the exception of PMA. Whereas PMA induced IL-6 production in endothelial cells and it potentiated the induction of IL-6 by IL-1 in these cells, it inhibited LPS-stimulated IL-6 production in monocytes. In line with the effects of PMA, staurosporin induced IL-6 production in monocytes and it inhibited IL-1 driven IL-6 production by endothelial cells.     

Abnormal NF-kappa B function characterizes human type 1 diabetes dendritic cells and monocytes

Dendritic cell (DC) differentiation is abnormal in type 1 diabetes mellitus (T1DM). However, the nature of the relationship between this abnormality and disease pathogenesis is unknown. We studied the LPS response in monocytes and monocyte-derived DCs isolated from T1DM patients and from non-T1DM controls. In T1DM patients, late LPS-mediated nuclear DNA binding by RelA, p50, c-Rel, and RelB was impaired as compared with type 2 DM, rheumatoid arthritis, and healthy subjects, associated with impaired DC CD40 and MHC class I induction but normal cytokine production. In TIDM monocytes, RelA and RelB were constitutively activated, and the src homology 2 domain-containing protein tyrosine phosphatase (SHP-1), a negative regulator of NF-kappaB, was overexpressed. Addition of sodium stibogluconate, a SHP-1 inhibitor, to DCs differentiating from monocyte precursors restored their capacity to respond to LPS in approximately 60% of patients. The monocyte and DC NF-kappaB response to LPS is thus a novel phenotypic and likely pathogenetic marker for human T1DM. SHP-1 is at least one NF-kappaB regulatory mechanism which might be induced as a result of abnormal inflammatory signaling responses in T1DM monocytes.     

Effect of interferon-alpha on immunoglobulin synthesis by human B cells

We have investigated the effect of human recombinant interferon-alpha (IFN-alpha) on mitogen-induced immunoglobulin (Ig) production by peripheral blood mononuclear cells from normal individuals. Low concentrations (1 to 100 IU/ml) of IFN-alpha enhanced pokeweed mitogen-stimulated Ig production. In contrast, high concentrations of IFN-alpha (10(5) IU/ml) suppressed pokeweed mitogen-induced Ig production. Irradiation of T cells did not ablate the high dose suppression, indicating that suppression was not due to a radiation-sensitive T cell. Kinetic experiments revealed that IFN-alpha needed to be added to 10 day cultures within the first 72 hr for either enhancement or suppression to be noted. Preincubation of purified B cells with IFN-alpha suppressed Ig production as completely as when unfractionated mononuclear cells were incubated with IFN-alpha. On the other hand, preincubation of T cells or monocytes with IFN-alpha had no effect on subsequent Ig production in reconstituted mononuclear cell cultures. Mitogen-induced proliferation of purified B cells was not affected by IFN-alpha at any concentration, but Ig production by purified B cells stimulated with Staphylococcus aureus Cowan I or anti-mu and B cell differentiation factors responded to IFN-alpha with low concentration enhancement and high concentration suppression. Studies of Ebstein-Barr virus-transformed B cell lines showed that IFN-alpha caused a similar effect on the CESS line as on peripheral blood B cells, with low dose enhancement and high dose suppression of Ig production. Thus one IFN-alpha effect is to modulate Ig production, and this appears to be a direct effect on B cells. Combined with the data in the accompanying paper, the effects of IFN-alpha on B cell function are similar in vivo and in vitro.