IL-2 production by B cells stimulated with a specific antigen

The ability of a specific antigen (Ag) to stimulate B cells to produce IL-2 was examined with a murine B lymphoma line, A20-HL, which expressed surface IgM specific for trinitrophenyl (TNP). The culture supernatant of A20-HL cells stimulated with TNP3.9-ovalbumin (-OVA) or anti-IgM goat IgG contained an activity which supported the proliferation of an IL-2-dependent T cell line, CTLL-2. Neither TNP3.9-OVA nor anti-IgM antibody stimulated the parent line, A20.2J, which did not bear TNP-specific sIg, whereas anti-mouse Ig rabbit IgG F(ab)2 did stimulate both A20-HL cells and A20.2J cells. The active material in the culture supernatant was identified as IL-2 based on the experiments in which the activity was inhibited by anti-IL-2 mAb, and IL-2 mRNA was expressed in A20-HL cells stimulated with TNP3.9-OVA or anti-IgM antibody. These results support the conclusion that a specific Ag can stimulate A20-HL cells to produce IL-2. For IL-2 production, TNP receptors on A20-HL cells have to be appropriately cross-linked, inasmuch as either TNP3.9-OVA or TNP6.7-OVA was much more effective than TNP1.2-OVA and TNP22.9-OVA in the induction of IL-2 production by A20-HL cells.     

Interleukin-4 enhances peritoneal B cell stimulation induced by phorbol ester

The activation requirements of murine peritoneal B cells differ from those of conventional (splenic) B cells; in particular, peritoneal B cells are stimulated to enter S phase by phorbol ester, acting alone. This pathway was studied to assess the susceptibility of peritoneal B cells to regulation by T cell products. Three T cell supernatants enhanced phorbol myristate acetate (PMA)-induced peritoneal B cell stimulation. This enhancement was reproduced by recombinant interleukin 4 (IL-4), and IL-4-mediated enhancement was reversed by 11B11 anti-IL-4 antibody. Enhancement of S phase entry was dose dependent for IL-4 and required stimulatory concentrations of PMA. In addition, IL-4 in combination with PMA produced a marked increase in IgM secretion by peritoneal B cells cultured in vitro. Neither an enhancement of S phase entry nor an increase in IgM secretion was observed with splenic B cells similarly treated with IL-4 and PMA. These results suggest that IL-4 modulates the proliferative and differentiative responses of the unusual B cells that reside in the peritoneal cavities of normal mice.     

Murine B lymphoma cell lines release functionally active interleukin 2 after stimulation with Staphylococcus aureus

In this study it is shown that an IL-2-like functional lymphokine activity derived from the murine B cell lines 2PK-3 and L10A2J after stimulation with Staphylococcus aureus can be blocked with anti-IL-2 mAb such as 9B11, DMS-1, and S4B6. Experiments demonstrate that the stimulation of the IL-2-sensitive cell line CTLL-2 by the IL-2-like functional activity derived from murine B cell tumors can also be blocked with the anti-IL-2R mAb PC61. Additionally, in RNA-RNA hybridization experiments with radiolabeled SP6-derived ssRNA probes developed from murine IL-2 genomic DNA sequences and specific for IL-2 mRNA, quantitatively significant amounts of IL-2-specific mRNA in both 2PK-3 and L10A2J are shown subsequent to stimulating the cells with S. aureus. These results suggest the murine B cell tumor lines 2PK-3 and L10A2J synthesize and release IL-2 after stimulation with selected polyclonal activators such as S. aureus.     

Stimulation of EBV-activated human B cells by monocytes and monocyte products. Role of IFN-beta 2/B cell stimulatory factor 2/IL-6

EBV infects human B lymphocytes and induces them to proliferate, to produce Ig, and to give rise to immortal cell lines. Although the mechanisms of B cell activation by EBV are largely unknown, the continuous proliferation of EBV-immortalized B cells is dependent, at least in part, upon autocrine growth factors produced by the same EBV-infected B cells. In the present studies we have examined the influence of monocytes on B cell activation by EBV and found that unlike peripheral blood T cells and B cells, monocytes enhance by as much as 30- to 50-fold virus-induced B cell proliferation and Ig production. Upon activation with LPS, monocytes secrete a growth factor activity that promotes both proliferation and Ig secretion in EBV-infected B cells and thus reproduces the effects of monocytes in these cultures. Unlike a number of other factors, rIFN-beta 2/B cell stimulatory factor 2 (BSF-2)/IL-6 stimulates the growth of human B cells activated by EBV in a manner similar to that induced by activated monocyte supernatants. In addition, an antiserum to IFN-beta that recognizes both IFN-beta 1 and IFN-beta 2 completely neutralizes the B cell growth factor activity of activated monocyte supernatants. These findings demonstrate that IFN-beta 2/BSF-2/IL-6 is a growth factor for human B cells activated by EBV and suggest that this molecule is responsible for B cell growth stimulation induced by activated monocyte supernatants. We have examined the possibility that IFN-beta 2/BSF-2/IL-6 might also be responsible for B cell growth stimulation by supernatants of EBV-immortalized B cells and thus may function as an autocrine growth factor. However, IFN-beta 2/BSF-2/IL-6 is not detectable in supernatants of EBV-immortalized B cells by immunoprecipitation. Also, an antiserum to IFN-beta that neutralizes IFN-beta 2/BSF-2/IL-6 fails to neutralize autocrine growth factor activity. This suggests that autocrine growth factors produced by EBV-immortalized B cells are distinct from IFN-beta 2/BSF-2/IL-6. Thus, the continuous proliferation of EBV-immortalized B cells is enhanced by either autocrine or paracrine growth factors. One of the mediators with paracrine growth factor activity is IFN-beta 2/BSF-2/IL-6.     

B cell stimulatory factor 1 (IL-4) enhances the development of cytotoxic T cells from Lyt-2+ resting murine T lymphocytes

B cell stimulatory factor 1 (BSF-1) (IL-4) was shown to synergize with phorbol esters or with monoclonal anti-TCR antibody in stimulation of the development of CTL from small resting murine T cells. IL-2 also synergized with PMA in such differentiation but was less effective than BSF-1. The combination of these two lymphokines with PMA had the most potent effect on the development of CTL. BSF-1 plus PMA stimulated a significant increase in the intracellular content of N-benzyloxycarbonyl-L-lysine thiobenzylester esterase, a granule-associated biochemical marker, whereas IL-2 plus PMA was only marginally effective. Depletion of L3T4+ cells did not result in the abrogation of these effects. Lyt-2+ T cells that were incubated for 72 h with BSF-1 plus PMA accumulated N-benzyloxycarbonyl-L-lysine thiobenzylester esterase and secreted this intragranular marker after interaction with immobilized anti-T cell receptor mAb. These BSF-1/PMA-stimulated Lyt-2+, L3T4- T cells were also able to kill FcR positive target cells in a retargeting assay with a mAb to murine T3 Ag, providing evidence that BSF-1 plus PMA acted directly on precursors of cytotoxic T cells.     

Role of B cell stimulatory factor 1/interleukin 4 in clonal proliferation of B cells

B cell stimulatory factor 1 (BSF-1)/interleukin 4 (IL-4) has striking effects on colony formation in soft agar by small resting B lymphocytes. BSF-1 alone induces colony formation in this cell population, presumably in costimulation with a mitogenic substance present in bacto-agar. In costimulation with anti-IgM antibodies, BSF-1 caused initial proliferation of 8 to 10% of B cells, resulting in a large number of cell clusters (10 to 40 cells/clone) after 3 to 4 days of incubation. However, substantial number of colonies (greater than 40 cells/clone) developed only from these clusters when IL-1 was added to the cultures. Using a modified immunoperoxidase staining technique for the determination of IgM allotype, evidence was obtained that B cell colonies stimulated with BSF-1 are derived from a single progenitor cell. Neutralization of BSF-1 with 11B11 after a culture period of 1 to 4 days inhibits further proliferation of B cell colonies, indicating that the action of BSF-1 extends for several cell generations beyond initial stimulation from the resting state. Furthermore, it is demonstrated that the synergistic action of IL-1 with BSF-1 is confined to the late culture period, indicating a growth-promoting effect by IL-1 for activated B cells.     

IL-4/B cell stimulatory factor 1 stimulates T cell growth by an IL-2-independent mechanism

Resting T cells are stimulated to synthesize DNA by IL-4 and phorbol myristate acetate (PMA). This response of T cells to IL-4 plus PMA is independent of the action of IL-2 as judged by 1) the lack of IL-2 in supernatants of stimulated cells, 2) the failure to detect IL-2 mRNA in stimulated cells by in situ hybridization, 3) the inability of anti-IL-2R antibody and of anti-IL-2 antibody to block responses to IL-4 plus PMA, and 4) the failure of cyclosporin A to block responses. T cells also respond to anti-CD3 antibodies and IL-4 in the presence of anti-IL-2R antibodies. IL-4 stimulation of growth of the long term T cell line HT-2 also appears to be independent of the action of IL-2. No IL-2 mRNA is found in IL-4-stimulated HT-2 cells by Northern blotting; the response of HT-2 cells to IL-4 is not blocked by anti-IL-2R antibodies; the response of HT-2 cells to IL-4 is not inhibited by cyclosporin A. Although IL-4 stimulation of T cells is independent of IL-2, IL-4 plus PMA treatment of resting T cells does cause enhanced expression of IL-2R and prepares cells to proliferate to IL-2 alone. In both these properties IL-4 resembles IL-2. These experiments lead us to conclude that IL-4 can act as an alternative to IL-2 as authentic T cell growth factor.     

Human B cell lines can be triggered to secrete an interleukin 2-like molecule

To determine whether human B cells can be triggered to secrete interleukin 2 (IL-2), 19 tumor cell lines derived from patients with undifferentiated lymphomas of Burkitt's and non-Burkitt's types and 6 normal lymphoblastoid cell lines were tested. Cells were grown in the presence or absence of the new tumor promoter teleocidin, and culture supernatants were assayed for IL-2 activity using the standard CTLL-2 assay. Teleocidin (10 ng/ml) triggered IL-2 secretion in 7/8 (87%) EBV-negative lymphoma cell lines of American origin and in 6/6 (100%) normal lymphoblastoid cell lines, but in only 1/6 (16%) EBV-positive tumor cell lines of American origin. Teleocidin had no effect on 5/5 (0%) African Burkitt's cell lines. IL-2 secretion was not detected in control supernatants. IL-2 secretion correlated with the induction of IgM secretion and was linked to both EBV status and karyotype. The following similarities in the functional biological characteristics of T cell and B cell IL-2 suggest that B cell IL-2 is not a factor which mimics IL-2 activity in the CTLL-2 assay: (i) neutralization of IL-2 by anti-IL-2 monoclonal antibody (DMS-1); (ii) elution of IL-2 following its adsorption to CTLL-2 cells; (iii) determination of the MW of IL-2 by SDS-PAGE and Western blot analysis; and (iv) ability of B cell IL-2 to support T cell proliferation and blocking of this activity by anti-tac monoclonal antibody. cDNA probes for T cell IL-2, however, did not detect IL-2 mRNA in B cells. The cell lines were also found to constitutively express IL-2 receptors detected by anti-tac monoclonal antibody, and to secrete soluble IL-2 receptors measured by ELISA. Our results imply that under certain circumstances, B cells can be triggered to secrete IL-2 or an IL-2-like molecule and thus influence T cell activation and proliferation.     

Interleukin-2 enhances the growth of human melanoma cells derived form primary but not from metastatic tumours

Previously, we demonstrated that in vitro treatment of B16F10 murine melanoma cells with interleukin-2 (IL-2) enhances proliferation and metastasis. To further investigate the role played by IL-2 in human melanomas, we studied the expression of IL-2/IL-2 receptor and the effect of IL-2 on the proliferation of melanoma cell lines derived from primary (A375 and RMS cell lines) and metastatic (Hs294T cell line) tumours. We found a constitutive expression of cytoplasmic IL-2 and alpha, beta and gamma-subunits of the IL-2R on the surface of the three melanoma cell lines. The presence of IL-2 in the culture increased the proliferation rate in A375 and RMS cell lines, but no effect was observed in Hs294T metastatic cells. Biologically active IL-2 could be found in the supernatant of the three melanoma cell lines, particularly in A375 and RMS cells, in which an inhibition of the proliferation rate was observed when IL-2 was blocked. Moreover, the combination of anti-IL-2R beta and anti-IL-2R gamma blocking antibodies induced a significant down-regulation of cell proliferation in the three melanoma cell lines, and the combination of anti-IL-2R alpha, anti-IL-2R beta and anti-IL-2R gamma blocking antibodies inhibited IL-2-mediated growth stimulation in A375 and Hs294T cell lines. In RMS cells, a more significant effect was observed when only IL-2R gamma was blocked. Finally, exogenous IL-2 modulated the IL-2 endogenously produced by melanoma cells. These data show that IL-2 may modulate the growth of melanoma cells through autocrine or/and paracrine mechanisms.     

T cell derived IL-6 is differentially required for antigen-specific antibody secretion by primary and secondary B cells

IL-6 (formerly PCTGF, HP-1, BSF-2, HGF, IFN-beta 2, 26 kDa) is a recently defined lymphokine demonstrating activity on multiple cell types, including hepatocytes, thymocytes, T cells, plasmacytomas, and B cells. The biologic effects of IL-6 on lymphocytes, particularly B cells, suggest this factor may be involved in the regulation of normal immune responses. Accordingly, we have investigated the role of IL-6 in Ag-specific responses of B cells from both naive and Ag-primed mice. When Ag-primed splenic T cells were used as a source of help, naive (primary) B cell responses specific for the hemagglutinin molecule of the influenza A virus (PR8) were fully inhibited by the addition of an anti-IL-6 antiserum, and are thus IL-6 dependent. In contrast, secondary B cell responses were essentially IL-6 independent, being unaffected by this antiserum even at concentrations 10-fold higher than required to completely inhibit primary responses. This differential IL-6 requirement was further investigated by using a panel of hemagglutinin molecule-specific Th clones. Consistent with the above findings, a Th1 clone secreting biologically active IL-6 enables antibody secretion by both primary and secondary B cells, whereas Th1 clones that do not produce IL-6 support secondary responses, but fail to help primary B cell responses unless exogenous IL-6 is added. These results provide the first instance of differential lymphokine requirements among primary vs secondary B cell responses, and suggest T cell-derived IL-6 plays a critical role during the regulation of humoral immune responses. Moreover, functionally distinct Th1 clones were identified that differed in IL-6 secretion and their corresponding ability to induce Ig secretion by primary and secondary B cells.     

Synergistic action of A23187 and phorbol ester on human B cell activation

We have investigated the existence of a synergy occurring between the calcium ionophore A23187 and phorbol myristic acetate (PMA) with respect to human B cell proliferation and differentiation. The combination of A23187 (250 to 500 nM) with nonmitogenic concentrations of PMA (1 to 3 ng/ml) resulted in a strong proliferative response in human tonsillar, spleen, and peripheral blood B cells. This proliferation could not be blocked by anti-Tac antibody at concentrations that effectively inhibited T cell proliferation under similar culture conditions, suggesting that IL 2 and its receptor are not involved in B cell proliferation in this system. During a 3-day culture period, A23187 (500 nM) did not activate B cells in terms of changes in cell size or in the expression of transferrin receptor, HLA-DR, and Tac antigen. PMA at a nonmitogenic concentration (3 ng/ml) enhanced the expression of the first two markers. Combination of the ionophore with PMA induced the occurrence of Tac and further increased the expression of transferrin receptor and HLA-DR. A23187 similarly enhanced the PMA-mediated increase in cell size. PMA and A23187 did not induce differentiation to lg production. However, when cells were prestimulated with a combination of the two agents and were recultured in the presence of a preparation containing B cell differentiation factor, a strong increase in IgM, IgG, and IgA production was found. We conclude that PMA and A23187 synergistically trigger intracellular events in human B cells, leading to proliferation and to responsiveness to differentiation factors.     

Adrenocorticotropic hormone controls Concanavalin A activation of rat lymphocytes by modulating IL-2 production

The initiation of DNA synthesis and secretion of Interleukin 2 (IL-2) was measured in isolated rat splenic lymphocytes following activation with Concanavalin A (ConA). The extent of 3H-thymidine incorporation into activated cells was tested when cultured with various concentrations of Adrenocorticotropic hormone (ACTH). A paradoxical dose-response curve resulted when ACTH caused a biphasic response of augmenting and inhibiting 3H-thymidine uptake in lymphocytes depending on the hormone concentration. Low levels of ACTH (0.001-1-nM) augmented 3H-thymidine uptake and high levels (10-1000 nM) reversed the effect. The optimal ACTH concentration was 10 pM ACTH in the presence of 5 ug/ml ConA and there was no ACTH effect on quiescent cells (no ConA). Conditioned media from splenic lymphocytes treated with various concentrations of ConA or ACTH was tested for increased uptake of 3H-thymidine by the IL-2 growth dependent Cytotoxic T Lymphocyte Leukemia (CTLL-2) cells. ConA conditioned medium could sustain the CTLL-2 cells indicating the presence of IL-2. Conditioned medium from splenic lymphocytes treated with both ConA and 100 pM ACTH further increased CTLL-2 cell proliferation indicating an additional increase of IL-2 secretion. The identity of IL-2 was confirmed by using an anti-rat IL-2 antibody to neutralize the growth potential of the conditioned medium. ACTH alone had no effect on the CTLL-2 cell proliferation indicating the effect is due solely to induced IL-2 found in the conditioned medium. IL-2 levels in the conditioned media were quantitated by ELISA assay; splenic lymphocytes produced 4.2 ng/ml to ConA only, 19.2 ng/ml in ConA plus 10 nM ACTH, and no detectable IL-2 at ConA plus 10 uM ACTH. These results demonstrated that ACTH modulates IL-2 secretion from activated lymphocytes, which is both biphasic and concentration dependent.     

Interleukin 2 produced by activated B lymphocytes acts as an autocrine proliferation-inducing lymphokine

Normal peripheral blood B cells produce a soluble factor after activation that is functionally indistinguishable from interleukin 2 (IL 2) and can support B cell proliferation in vitro. Purified rabbit peripheral blood B cells, when stimulated with a combination of ionomycin (0.5 microgram/mL) and phorbol myristate acetate (PMA) (1 ng/mL), secreted a soluble factor in the culture medium that supported the IL 2-dependent cell line CTLL-2. The ability of these supernatants to support CTLL-2 growth was almost completely blocked by rabbit antibodies against human recombinant IL 2 and by the anti-IL 2 receptor monoclonal antibody 7D4. These data strongly suggest that the growth factor secreted by rabbit B cells is IL 2. To examine the possibility that the IL 2 activity detected in the B-cell cultures may be derived from residual T cells, B cells were further purified by successive panning with a pan-T-cell monoclonal antibody, L11-135, and goat anti-rabbit IgG. These highly purified B cells produced levels of IL 2 activity comparable to those produced by the initial B cell populations. Comparison of IL 2 production by decreasing numbers of purified T cells and purified B cells also indicated that the B cells were the source of IL 2 activity. Supernatants of activated B cells could support proliferation of B-cell blasts, and this activity could be completely absorbed by CTLL-2 cells, indicating that IL 2 is a major growth factor for B cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relative roles of T-cell receptor ligands and interleukin-2 in driving T-cell proliferation

Stimulation of T cells by the T-cell receptor (TCR)/CD3 complex results in interleukin-2 (IL-2) synthesis and surface expression of the IL-2 receptor (IL-2R), which in turn drive T-cell proliferation. However, the significance of the requirement of IL-2 in driving T-cell proliferation, when TCR stimulation itself delivers potential mitogenic signals, is unclear. We show that blocking of IL-2 synthesis by Cyclosporin A (CsA) suppressed both the Concanavalin A (Con A)- and phorbol myristate acetate (PMA)/ionomycin-induced proliferation of T cells. The latter is also inhibited by anti-IL-2R. Kinetic studies showed that T-cell proliferation begins to become resistant to CsA inhibition by about 12 h and became largely resistant by 18 h of stimulation. PMA, the protein kinase C activator, enhanced Con A-induced T-cell proliferation if added only within first 12 h of stimulation, and not after that. Given the fact that, in the present study, TCR is downregulated within 2 h of Con A stimulation and T cells entered the S phase of cell cycle by about 18 h of stimulation, the above results suggest that TCR stimulation provides the initial trigger to the resting T cells, which allows the cells to traverse the first two third portions of G1 phase of cell cycle and become proliferation competent. IL-2 action begins afterward, delivering the actual proliferation signal(s), allowing the cells to traverse the rest of G1 phase and enter the S phase of the cell cycle.     

The requirement for high intracellular cyclic adenosine monophosphate concentrations distinguishes two pathways of B cell activation induced with lymphokines and antibody to immunoglobulin

Mouse B lymphocytes proliferate when stimulated with anti-IgM and the lymphokines B cell-stimulating factor-1 (BSF-1) interleukin (IL)-4 and IL-1. We show herein that two anti-IgM-stimulated B cell responses can be distinguished by their sensitivity to cyclic adenosine monophosphate (cAMP). cAMP inhibits B cell proliferation facilitated by BSF-1 and enhances the B cell response facilitated by IL-1. IL-1 and BSF-1-dependent B cell responses are additive, suggesting that distinct B cell subpopulations are involved. BSF-1-reactive B cells are not inhibited by cAMP in the presence of IL-1. This suggests that IL-1 determines whether cAMP, which has elsewhere been shown to cause nuclear translocation of protein kinase C in resting B cells, inhibits or enhances B cell responses. Comparative studies suggest that the IgM response of spleen cells to red cell-bound antigen involves one of the two pathways described, namely the one that involves IL-1 and cAMP. BSF-1 has no noticeable effect on a Mishell-Dutton plaque-forming cell response. In view of literature on the facilitation by BSF-1 of the IgG response, we consider the possibility that IL-1 functions to stir B cells into an IgM response, whereas BSF-1 acts on B cells that are poised to become IgG producers.     

Protein kinase C-dependent and -independent mechanisms of cloned murine T cell proliferation. The role of protein kinase C translocation and protein kinase C activity

PMA can induce the proliferation of several CTL clones but not of several Th clones derived and tested in our laboratory. The PMA-stimulated proliferation of our CTL clones (which do not make IL-2 mRNA or protein) occurs independently of IL-2 and is not accompanied by lymphokine release. We now report, however, that protein kinase C (PKC) translocation is induced by PMA in CTL clones as well as in Th clones, which lack a proliferative response to PMA. These results suggest that PKC translocation itself is not a sufficient regulatory mechanism to account for cloned T cell proliferation. Moreover, IL-2 did not induce PKC translocation in a CTL clone, which proliferates when stimulated with IL-2. Thus, PKC translocation may not be necessary for activation of CTL proliferation. Nonetheless, cellular PKC activity appears to be required for the proliferative response of T cell clones after stimulation by PMA/PMA + calcium ionophore (A23187) or by triggering through the TCR: chronic PMA treatment, which depletes intracellular PKC activity, abrogates the proliferative response of T cell clones stimulated by PMA/PMA + A23187 or triggered through the TCR. T cell clones depleted of PKC activity, however, retain the ability to proliferate when challenged with IL-2. Murine T cell clones, therefore, possess PKC-dependent and PKC-independent pathways of proliferation that are not regulated by PKC translocation alone.     

Mechanism of T cell proliferation in vivo: analysis of IL-2 receptor expression and activation of c-myc and c-myb oncogenes during lymphatic regeneration

The mechanism of T cell proliferation was studied using in vivo lymphatic regeneration as the model. Lymphatic regeneration was induced by injecting a sublethal dose (300 mg/kg) of cyclophosphamide (Cy) into mice. Majority of the regenerating splenic T cells were found to be in the cell cycle, nearly 30% being found in S/G2+M phases resembling the ratio obtained for mitogen activated T cells in vitro. Expression of interleukin-2 receptor (IL-2R) was defined by the monoclonal anti-IL-2R antibody, AMT-13. Only 1-3% of regenerating T cells were IL-2R positive (while about 30% of the in vitro activated T cells were IL-2R positive). Accordingly, these cells did not respond to IL-2 in vitro. However, when the freshly isolated regenerating T cells were cultured in the presence of Con A or PMA + ionophore A 23187, IL-2R was readily induced. The regenerating T cells were further analyzed for the expression of the cellular oncogenes c-myc and c-myb. These cells expressed about three times more c-myb mRNA than Con A-stimulated T cells and the levels were comparable to those seen in thymocytes. By contrast, the amount of c-myc mRNA was similar in the regenerating T cells and in Con A-activated T cells, but weak or barely detectable in splenocytes and thymocytes. Taken together, our results imply that the vigorous T cell proliferation during cyclophosphamide-induced lymphatic regeneration is independent of the IL-2/IL-2R hormone system, like T-cell precursor proliferation in the thymus, and is characterized by both high c-myb expression typical for thymocytes and high c-myc expression typical for in vitro proliferation-activated T cells.     

Regulation of BSF-2/IL-6 production by human mononuclear cells. Macrophage-dependent synthesis of BSF-2/IL-6 by T cells

Regulation of BSF-2/IL-6 production in peripheral mononuclear cells (MNC) was studied. BSF-2 mRNA expression in mitogen-stimulated MNC showed a biphasic response, the first peak around 4 h and the second peak around 48 h. This was caused by different kinetics of BSF-2 mRNA expression in distinct subpopulations of MNC; M phi expressed BSF-2 mRNA at 5 h in the absence of any stimulation, and mitogen-stimulated T cells and B cells expressed BSF-2 mRNA 48 h after stimulation. Immunohistochemical staining of the cells with anti-BSF-2 antibody demonstrated that macrophages, T cells and B cells could produce BSF-2. T cells in peripheral MNC produced BSF-2 in the presence of M phi. The requirement of macrophage for BSF-2 production in T cells could be replaced by TPA but not by IL-1 or BSF-2.     

IL-4 promotes the migration of circulating B cells to the spleen and increases splenic B cell survival

We report that IL-4 causes a redistribution of B cells and modestly increases B cell life span. Intravenous injection of a long-acting formulation of IL-4 induces increases in both spleen cell number and the percentage of splenic B cells. These effects are observed within 1 day of IL-4 administration and plateau after approximately 3 days if IL-4 treatment is continued. The increase in splenic B cell number is IL-4 dose dependent, CD4+ T cell independent, FcgammaRII/FcgammaRIII independent, and Stat6 independent. Decreases in the number of B cells in the blood and the percentage of mature B cells in the bone marrow, concomitant with the increase in splenic B cell number, suggest that redistribution of circulating B cells to the spleen is partially responsible for IL-4 induction of splenic B cell hyperplasia. Considerable reduction in the effect of 5 days of IL-4 treatment on splenic B cell number when B lymphopoiesis is blocked with anti-IL-7 mAb suggests that generation of new B cells is also involved in IL-4-induced splenic B cell hyperplasia. 5-Bromo-2'-deoxyuridine labeling experiments demonstrate that IL-4 modestly prolongs the life span of newly generated splenic B cells, and experiments that measure B cell HSA (CD24) expression as an indicator of B cell age suggest that IL-4 may also prolong the life span of mature splenic B cells. Thus, IL-4 increases splenic B cell number through two Stat6-independent effects: increased net migration of circulating B cells to the spleen and increased B cell life span. Both effects may promote Ab responses to a systemic Ag challenge.