A new synthesis of cytoxazone and its diastereomers provides key initial SAR information.

A short, enantioselective, and diastereoselective synthesis of cytoxazone, a Th2-selective immunomodulator from Streptomyces, is described. The route was readily adapted to the synthesis of the three other stereoisomers of natural cytoxazone. Evaluation of these compounds revealed that the stereochemical configuration of the oxazolidinone ring did not influence their biological activity.

Resistance to PGE2 inhibition of PWM-stimulated lymphocytes from neoplastic patients.

PGE2 treatment of mononuclear cells from patients with different types of neoplasias was unable to decrease either the number of plaque-forming cells or the expression of CD71 and CD25 in PWM-driven cultures. In contrast, in previous studies, PGE2 inhibited these parameters in cultured mononuclear cells from normal volunteers. Surgical treatment of cancer patients did not modify the lymphocyte sensitivity to PGE2 after 1 week, but at 2 and 6 months after therapeutical treatment, the inhibition values of the parameters studied were almost similar or very similar to those of normal lymphocytes. The reduction of PGE2 sensitivity in cancer patients was related to the increase of PGE2 levels and, probably, to a PGE2 receptor saturation. A restoration of PGE2-induced inhibition some months after therapy could be due to the decrease in PGE2 levels and to receptor unsaturation.

Induction of inhibitory activity for B cell differentiation in human CD8 T cells with pokeweed mitogen, dimaprit, and cAMP upregulating agents: countersuppressive effect of platelet factor 4.

As shown previously, native or recombinant (r) human platelet factor 4 (PF4) alleviates the suppression induced by Con A or dimaprit, a histamine type 2 receptor (H2-R) agonist, in a murine system. The effect of rPF4 on human peripheral blood cells has now been studied, using as a model pokeweed mitogen (PWM)-induced, T-cell-mediated suppression of Ig-secreting cell (ISC) formation by Staphylococcus aureus and rIL-2 activated B cells. PWM, but not phytohemagglutinin (PHA), induced inhibitory activity in mitomycin-treated CD8+ T cells, but not unfractionated or CD4+ T cells, for both ISC formation and B cell proliferation. rPF4 and its C-terminal tridecapeptide alleviated the suppressive effect of PWM-activated CD8+ T cells on ISC production but not on proliferation. Heparin did not prevent this immunoregulatory activity of PF4. Neutralizing antibody to TGF-beta, but not to IFN-gamma or TNF-alpha, alleviated the suppression of ISC formation in some of the experiments. The H2-R appeared to play a part in inducing suppression, because the H2-R antagonist, cimetidine, prevented the PWM-induced suppression of ISC production. Furthermore, dimaprit induced suppression of ISC formation when added instead of PWM at the start of culture. Incubation of CD8+ T cells with dimaprit for only 3 hr prior to coculture with S. aureus + IL-2 activated B cells decreased the ISC response. This suppression was also alleviated by addition of rPF4 to the coculture. Similar to dimaprit, known cAMP upregulating agents, such as forskolin, dibutyryl cAMP, and cAMP analog, all induced this immunoregulatory activity in T cells. Moreover, the effect of dimaprit was prevented by the specific protein kinase A inhibitor, HA1004, suggesting strongly that upregulation of cAMP played a role in the H2-R-mediated effect. Cell contact appeared to be necessary, since supernatants from dimaprit or PWM activated T cells failed to suppress ISC production. We suggest that the known ability of PF4 to prevent TGF-beta-mediated effects on endothelial and other target cells may be involved in the alleviating effect of PF4 on the cell-contact-dependent CD8+ T-cell-mediated B cell suppression.

Decreased levels of TNF-beta in cultured PBLs from HIV+ patients occur concomitantly to increased apoptosis and impaired PWM-induced proliferation and dehydrogenase activity.

The occurrence of apoptosis in cultured blood cells from HIV+ patients is well-documented. However, the relationship of this process to cytokine production is still undefined. We measured the production of TNF-beta by mitogen-stimulated PBLs from 33 HIV+ patients, while simultaneously assessing their development of apoptosis, dehydrogenase activity and proliferative responses to PWM and PHA; Only 3/33 patients had less than 30% apoptosis in PWM cultures (average value = 19.6%); patients were grouped in accordance with their having low (31-40%; average 34.8%), intermediate (41-50%, average 45.7%) or high levels (over 51%, average 53.6%) of apoptosis. Our results indicate that there is a quantitative correlation between the different degrees of apoptosis and the impaired production of TNF-beta, and support the hypothesis that PBLs from all HIV+ patients have defective immunological functions. In addition, our results show that TNF-beta production correlates with a stage classification of patients which reflects their PBL status of apoptosis, proliferative response to PWM and dehydrogenase activity in vitro.

Immunoglobulin binding factor in human seminal plasma: immunological function.

Human seminal plasma contains a protein with an estimated molecular weight of 16 kd that binds serum immunoglobulin gamma (IgG) and is named IgG binding factor (IgBF). Purified IgBF specifically suppressed pokeweed mitogen-induced lymphocyte blastogenesis, having little or no effect on lymphocyte blastogenesis stimulated with phytohemagglutinin or Concanavalin A; antibody-dependent cell-mediated cytotoxicity; natural killer cell activity; or complement-dependent cytotoxicity of antibodies against sperm. It would appear that IgBF may suppress activation of B cells in the male and female genital tract.

1,25 dihydroxyvitamin-D3 regulation of immunoglobulin production in peripheral blood mononuclear cells of patients with systemic lupus erythematosus.

Systemic Lupus Erythematosus (SLE) is a multisystem disease characterized by an increase in the secretion of autoantibodies. The mechanisms of autoantibody-induced disease have not been clarified. 1,25-Dihydroxy-vitamin D3 (1,25-D3) is known to be important in the regulation of normal human lymphocyte functions. Among its regulatory functions is the ability to inhibit mitogen-stimulated production of immunoglobulin. The experiments reported here explored the regulation of IgG production by peripheral blood mononuclear cells (PBMCs) of patients with inactive and active SLE. 1,25-Dihydroxyvitamin D3 inhibited mitogen-stimulated IgG production in cells from normal individuals and inactive SLE patients, but not spontaneous IgG production by PBMCs from active SLE patients. Addition of exogenous IL-2 (5-50 U/ml) to 1,25-D3-treated cells from all patient groups did not affect IgG production significantly under any conditions tested. The addition of IL-2 to PMBCs had no effect on IgG production in normal individuals or inactive SLE patients, but stimulated IgG production in PBMCs of active SLE patients. We conclude that the regulation of mitogen-stimulated IgG production in inactive SLE patients by 1,25-D3 and IL-2 is similar to normal individuals, but IgG production by active SLE PBMCs is unresponsive to 1,25-D3 regulation and is increased with the addition of IL-2.

Suppression of lymphocyte mitogenesis by tamoxifen.

Tamoxifen (at 0.1-1 microM concentrations) inhibited significantly the response of rat spleen cells to phytohemagglutinin, concanavalin A, pokeweed mitogen and lipid A in a dose-dependent manner. To achieve this inhibition, it was sufficient to expose the lymphocytes for 4 h to tamoxifen prior to mitogenic stimulation. Estradiol did not have a consistent effect on lymphocyte mitogenesis under the same conditions and did not modify the suppressive effect of tamoxifen, even when the lymphocytes were treated first with estradiol followed by tamoxifen. It is suggested that the inhibitory effect of tamoxifen on these in vitro lymphocyte reactions is not mediated by the estrogen receptor.

Zinc inhibits pokeweed mitogen-induced development of immunoglobulin-secreting cells through augmentation of both CD4 and CD8 cells.

The mechanism whereby zinc regulates the in vitro antibody synthesis of human peripheral blood mononuclear cells was investigated. In a serum-free culture, zinc inhibited the pokeweed mitogen (PWM)-induced generation of immunoglobulin secreting cells (ISC) through an apparent non-specific augmentation of T-cells, which included CD4 and CD8 cells, as well as both CD8-Leu8+ and CD8-Leu8- cells. It was noted that CD4 cells were more stimulated than CD8 cells, and that CD8-Leu8- cells were more stimulated than CD8 Leu8+ cells. CD8-Leu8- cells inhibited the development of ISC. These findings indicate that, although zinc stimulates all T-cells, the relative potency of zinc differs among T-cell subsets; helper inducer T-cells appear to be most strongly stimulated.

Anti-pokeweed mitogen antiserum inhibits and enhances blastogenesis of mononuclear cells induced by pokeweed mitogen.

The interaction between pokeweed mitogen (PWM) and peripheral blood mononuclear cells (PBMC) was investigated using rabbit anti-PWM antiserum (anti-PWM) and 125I-PWM. Incubation of PBMC with PWM in the presence of anti-PWM resulted in an inhibition of the mitogenic effect of PWM. Anti-PWM predominantly blocked the interaction of PWM with monocytes, which is essential for optimal stimulation of lymphoid cells with PWM. Addition of anti-PWM to PBMC at several time-points after incubation with PWM showed inhibition of mitogenic activity when anti-PWM was added within 8 hours. However, enhancement of PWM-induced blast cell formation was found when anti-PWM was added after 48 hours. Further analysis revealed that the inhibition of PWM stimulation was mediated by the F(ab')2 part of anti-PWM IgG. On the other hand F(ab')2-anti-PWM was not able to enhance the effect of PWM. Incubation of PBMC with 125I-PWM and anti-PWM simultaneously, decreased the binding of PWM to both lymphocytes and monocytes. In contrast, addition of anti-PWM 48 hours after the incubation of PBMC with PWM resulted in an increased binding of PWM to monocytes. These results show that anti-PWM can modulate the lymphocyte reaction to PWM and suggest two possible mechanisms by which PWM can stimulate PBMC, both of which are dependent on the interaction of PWM with monocytes.

CD3 modulation inhibits pokeweed mitogen-induced T-cell help for immunoglobulin production.

The CD3 molecule is considered to be a signal transducer in the process of T-cell activation. Modulation of the CD3 molecule of peripheral blood T cells can be accomplished by incubation at 37 degrees C with UCHT-1, a mouse IgG1 anti-CD3 monoclonal antibody, under experimental conditions avoiding T-cell activation. We have examined the effect of CD3 modulation on T-cell-dependent polyclonal immunoglobulin (Ig) production induced by pokeweed mitogen (PWM) in cultures of peripheral blood lymphocytes. CD3 modulation strongly inhibited (greater than 80%) IgG and IgM production. This was due to inhibition of the production of soluble helper factors by the T cells, and not to induction of suppressor cells. These data support the concept that the CD3 molecule is an essential signal transducer in the process of PWM-induced helper T-cell activity, and that CD3 can function as a receptor transmitting negative signals to helper T cells.

Cyclosporin A directly inhibits human B-cell proliferation by more than a single mechanism.

Cyclosporin A (CsA) is a potent immunosuppressive agent that inhibits T-cell proliferation and lymphokine production. There is less information on the direct effect of CsA on B-cells. We investigated the proliferative responses of human tonsillar B-lymphocytes to a "T dependent" mitogen, pokeweed mitogen (PWM), and to a "T independent" mitogen, Staphylococcus aureus (SA). Both responses were strongly inhibited by CsA. Nonspecific cytotoxicity was ruled out, and the inhibition was not reversed by adding IL1, IL2, or BCGF individually or in combination. Maximal inhibition of the PWM response occurred when CsA was added early in the culture period. Cyclosporin A added 18 hours after the start of culture was less effective, and adding CsA after 36 hours resulted in only minimal inhibition. However, with SA as mitogen, addition after 36 hours still affected substantial inhibition. These results, on the time of action and resistance to reversal by exogenous growth factors, suggest that CsA can directly inhibit human B-cells by a mechanism similar to its action on T-lymphocytes, blocking an early event critical to entry into cell cycle, but an additional mechanism of inhibition later in the cell cycle may also operate when the proliferative signal is provided by the T-independent mitogen SA.

Carrageenan-induced suppression of chicken lymphocyte proliferation.

Mitogenic responsiveness of peripheral blood lymphocytes (PBL) of chickens was suppressed by either pretreatment with or addition to the culture medium of various concentrations of carrageenan (CGN). Pretreatment for 1 hr significantly suppressed response to Concanavalin A (Con A) and Pokeweed mitogen (PWM) but did not affect Phytohemagglutinin (PHA) induced stimulation. Extension of the pretreatment period to 4 hrs suppressed response induced by all three mitogens. On the other hand, addition of carrageenan to the culture medium caused a dose-dependent suppression of PHA and Con A mediated response, but the effect on stimulation due to PWM was equivocal. In addition, low concentrations of CGN were weakly mitogenic to PBL and splenic lymphocytes.

Characterization of a soluble suppressor of human B cell function produced by a continuous human suppressor T cell line. II. Evidence for suppression through a direct action of CTC-SISS-B on human B cells.

CTC-SISS-B is an antigen-nonspecific suppressive lymphokine elaborated by an interleukin 2-dependent suppressor T cell line that produces noncytotoxic inhibition of human B cell but not T cell function. Like SISS-B, a soluble suppressive lymphokine present in the supernatants of Con A-activated peripheral blood T cell cultures, CTC-SISS-B is of 60,000 to 90,000 m.w., and its action is blocked by the simple sugar L-rhamnose. CTC-SISS-B inhibits human B cell Ig production and proliferation through a direct interaction with human B cells rather than through indirect effects on immunoregulatory T cells or monocytes. CTC-SISS-B suppression occurs through inhibition of an early event(s) in B cell activation since proliferation and Ig production by established human B cell lines are not inhibited by this lymphokine. Despite sharing many biochemical and biologic properties, CTC-SISS-B and gamma-interferon appear to be distinct mediators.

Interferon inhibits PWM induced B cell differentiation but not onset of proliferation.

The dependence of B lymphocyte differentiation into plasmacytes on anteceding B and T cell proliferation was studied using interferon as a probe. Possible correlations of the effect of interferon on PWM induced T and B cell proliferation and B cell differentiation into either kappa or lambda light chain immunoglobulin synthesizing plasmacytes have been investigated. The hypothesis that the observed inhibition of the PWM induced formation of plasmacytes by interferon is due to putative enhanced suppressor cell activity resulting from increased T cell proliferation is tested. Human, peripheral blood lymphocytes were exposed to PWM in the presence or absence of human leukocyte interferon. Proliferation was assayed by pulse cytophotometric analysis of cell kinetics, as well as [3H]TdR labelling of S-phase cells. Incidence of plasmacytes was detected by immunofluorescence using kappa or lambda light chain specific antibody. During continuous [3H]TdR labelling of stimulated cells, interferon inhibited incorporation of precipitable label by 40% at 96 and 144 h, indicating reduced net DNA synthesis by interferon treated cells. The relative fraction of cells in S-phase as well as G1- and G2 + M- was similar for treated and untreated cells. The fraction of cells rosetting SRBC remained stable for both treated and untreated cells. The size of the interferon treated population was persistently smaller once proliferation began. The time of initiation of proliferation was comparable for treated and untreated cells. Consistent with the findings of others using cell lines, interferon apparently induces a dilation of all cell cycle phases, thereby reducing the rate of proliferation. The same reduction occurred for both T and B cells. Time of initiation of DNA synthesis was, in contrast, not delayed by interferon, suggesting it is specific for events during the proliferative cell cycle. The occurrence of both kappa and lambda light chain immunoglobulin secreting plasmacytes was inhibited by interferon. The degree of inhibition was comparable for both kinds of plasmacytes detected. While not delaying the onset of DNA synthesis, interferon apparently retards subsequent cell proliferation and inhibits the differentiation of B cells to plasmacytes. The data indicate that active cellular proliferation and B cell differentiation require interferon sensitive events which cells initially recruited from quiescence by PWM do not. The inhibition of the incidence of plasmacytes cannot be attributed to an imbalance of T cell proliferation relative to non-T cells.