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Previous studies have shown that a flavonoid-phenolic-rich glycoprotein (TGP) is a mitogen to murine B cells. To test the hypothesis that this could be duplicated by a protein conjugate of the phenolic moiety, the response to rutin (R)-BSA was studied. It was demonstrated that: (I) R-BSA, like TGP, activates mouse B cells to proliferate; (ii) the kinetics of proliferation induced by R-BSA and TGP are similar; (iii) the mitogenic effects of neither R-BSA nor TGP are inhibitable by free flavonoids or phenolics; (iv) there is no age-dependent decrease in the proliferative response to either R-BSA or TGP; (v) both R-BSA and TGP induce spleen cells to secrete IL-6 by 2 hr of culture. They differ in that the proliferative response of congenitally athymic mice to R-BSA, but not to TGP, is significantly lower than that of euthymic mice, and in that TGP seems to stimulate a small subpopulation of T cells to enter cell cycle. The importance of the phenolic moiety in the response to TGP is supported by the observation that R-BSA immunoprecipitates cell surface components that seem to be identical to those precipitated by TGP. The apparent molecular sizes of the bands are approximately 110, 70, 55, 43, 34, 29, and 25-23 kDa. The 2-D analyses of the TGP and R-BSA precipitates are also striking in their similarity. The isoelectric point (pI) of the 28-kDa band is between pH 6.3 to 6.6. A band at approximately 23 kDa has a pI of 6.0, one band at approximately 25 kDa has a pI of approximately 5.4, and five bands ranging in size from approximately 26 to approximately 110 kDa all have a pI in the pH range of 4.6 to 5.4. The presence of multiple binding sites suggests that these compounds might activate cells via multiple distinct pathways.

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To test the hypothesis that inflammatory cytokine production might be an early event in the development of the disease associated with smoking, we used alveolar cells from healthy nonsmokers stimulated with TGP as a model system. TGP, a phenol-rich glycoprotein which is present in tobacco leaves and cigarette smoke condensate, activates the immune system. It stimulates polyclonal B cell differentiation, induces primarily an IgE response, and activates human leukocytes to produce IL-1. Using in situ nucleic acid hybridization we show that the steady-state levels of IL-1 alpha, IL-1 beta, IL-6, platelet-derived growth factor (PDGF)-A, and PDGF-B mRNAs are consistently elevated in the alveolar cells of all donors following TGP stimulation. The kinetics of mRNA expression suggest that IL-1 alpha and IL-1 beta mRNAs are independently regulated in alveolar cells, while the regulation of PDGF-A and PDGF-B mRNA seems to be similar. The activated cells also synthesize elevated levels of IL-1 and IL-6. These findings lend support to the suggestion that some clinical consequences of smoking might be initiated and enhanced by the production of inflammatory cytokines. Moreover, IL-6 could also activate a polyclonal B cell response, which could lead to the synthesis of autoantibodies and thus cause immune-mediated tissue injury.

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The secondary immune response classically differs from the primary response in magnitude, avidity, and isotype of the antibodies produced. Cell transfer studies to assess the contribution of memory B and memory T cells to each of these parameters are described. Avidities of the anti-DNP plaque-forming cells (PFC) generated in lethally irradiated recipients of naive B cells and keyhole limpet hemocyanin (KLH)-primed T cells, followed by immunization with soluble DNP-KLH, are medium to high, and do not differ significantly from the avidities of anti-DNP PFC in recipients of DNP-primed B cells and KLH-primed T cells. However, the number of indirect (I)-PFC and the ratio of I-PFC to direct (D)-PFC are significantly greater in the recipients of primed B and primed T cells. The results suggest that carrier primed T cells can selectively activate virgin B cells which are committed to produce medium- and high-avidity antibodies, and/or enhance the generation of somatic mutation which leads to antibodies of higher avidity. Priming of B cells is necessary for the increased magnitude of the I-PFC.

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The polyphenol group rutin (R) appears to influence isotype expression, because R-BSA conjugates induce anti-BSA responses in mice that show a significant decrease in hemagglutinating antibodies (HA) to BSA, as compared to mice immunized with BSA. However, the level of IgE antibodies to BSA is unaltered. To determine if suppressor cells for isotypes other than IgE are induced by R-BSA, cell transfers were performed. The results were consistent with the view that the decrease in HA titer to BSA in R-BSA immunized mice is not due to the activation of suppressor cells for isotypes other than IgE. Inasmuch as the IgE response in mice is associated with the production of IL-4 by Th2 cells, we analyzed the factors produced by spleen cells cultured with R-BSA. We found that supernatant from spleen cells cultured with R-BSA contained IL-4 as determined by the enhanced expression of Fc epsilon R (CD23) on B cells. This enhancement was inhibited by 11B11, the anti-IL-4 mAb. IL-2, a product of Th1 cells, was not detected in these supernatants. Moreover, IL-4 mRNA, but not IL-2 mRNA, was detected by Northern blot analysis of RNA from spleen cells cultured with R-BSA. Taken together the data suggest that the polyphenol containing compounds preferentially activate Th2 cells, thereby favoring IgE production.

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The regulation of human chorionic gonadotropin (hCG) secretion by placental trophoblasts is incompletely understood. A recent study reports that Interleukin-1 beta (IL-1 beta) stimulates hCG production in vitro by human, first trimester, placental trophoblasts, but not by a human choriocarcinoma cell line. Human decidua has been shown to produce IL-1 alpha and beta, and Tumor Necrosis Factor alpha (TNF alpha). The precise role(s) of these proteins in pregnancy is unknown. In the present study, hCG production by human choriocarcinoma cells (JAR) was evaluated in the presence of recombinant human IL-1 alpha (rHIL-1 alpha) and rHTNF alpha. hCG production was increased by rHIL-1 alpha in a dose-dependent manner, and heat-inactivation of this cytokine abolished the effect. Equimolar quantities of rHTNF alpha failed to influence hCG production or cell viability. IL-1 may be important in the regulation of hCG production by human trophoblasts, and therefore may play a physiologic role in pregnancy. Furthermore, TNF does not appear to participate in the regulation of the production of this hormone by human choriocarcinoma cells. This is the first demonstration of a divergence of activity of these two cytokines in the reproductive process.

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Human diseases caused by the intracellular bacterium Chlamydia trachomatis include genital tract infections and blinding trachoma. Chlamydial infections are characterized by chronic inflammation and scarring, and development of such complications is thought to be immunologically mediated. In this study, we show that coculture of C. trachomatis (serovar L2) with human blood monocytes induced the production of interleukin-1 (IL-1), an important mediator of inflammation, tissue remodeling, and scarring. IL-1 was produced in response to UV-inactivated elementary bodies containing from 0.1 to 50 micrograms of protein per ml, with a maximal response at 5 to 10 micrograms/ml. IL-1 activity was detected by 6 h of incubation and was maximal by 24 h. Peak levels were maintained throughout 96 h of incubation. Rabbit antibody to human IL-1(alpha + beta) effectively neutralized the thymocyte-stimulating activity of the supernatants. The apparent molecular weight of chlamydia-induced IL-1 was 16,000, as determined by gel filtration on a Bio-Gel P-60 column. Isoelectric focusing yielded two peaks of activity, with pIs of 5.5 and 6.9. Neutralization studies with antisera against human IL-1 alpha and IL-1 beta showed that the acidic and neutral peaks corresponded to IL-1 alpha and IL-1 beta, respectively, with IL-1 beta predominating. Heat-killed chlamydiae, which are not internalized by monocytes, were effective IL-1 inducers, indicating that phagocytosis was not required for IL-1 induction. Purified C. trachomatis lipopolysaccharide was also an effective IL-1 inducer, suggesting that the response to intact organisms may be largely a response to chlamydial lipopolysaccharide. Finally, purified chlamydial major outer membrane protein induced low but detectable IL-1 activity.

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Bacterial endotoxins (LPS) causes placental injury and fetal demise in pregnant animals. Because several biological effects of LPS are mediated by interleukin-1 (IL-1) and tumor necrosis factor (TNF), the hypothesis that these cytokines could cause placental injury similar to that seen in LPS-treated pregnant rats was tested. On day 12 of gestation, rats were injected intraperitoneally with saline, LPS, native or heat-inactivated (HI) rHIL 1 alpha, or rH-TNF alpha. Seven days later, grossly abnormal implantation sites and fetal demise were observed in rats injected with rHIL-1, rHTNF, or LPS but not in those injected with saline or HI-cytokines. Necrosis of placental, decidual, and fetal tissues was observed in cytokine-treated animals. The necrosis was more severe in LPS-treated rats, in which no fetal remains were identifiable. These data suggest that IL-1 and TNF may play a role in the fetoplacental injury observed in LPS-treated pregnant rats.

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We have previously shown that tobacco glycoprotein (TGP), a polyphenol-rich glycoprotein isolated from tobacco or from cigarette smoke, affects the immune system. In this study we show that TGP induces human PBL and adherent cells to produce IL-1 alpha and IL-1 beta. Two peaks of IL-1 activity were observed; one at 18-24 h, the second at 4-6 d after initiation of culture. A similar pattern was observed for the steady state level of IL-1 mRNA. These data suggest that the production of IL-1 by cells stimulated with TGP might be a factor in cardiovascular disease associated with cigarette smoking.

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We have been studying the effects of tobacco glycoprotein (TGP), a polyphenol-rich glycoprotein isolated from cured tobacco leaves, on the immune system. We have shown previously that mice immunized with TGP produce preferentially antibodies of the IgE isotype and that TGP is a T cell-independent B cell mitogen for mice, which stimulates B cell proliferation and B cell differentiation into Ig-secreting cells. We report herein that TGP stimulates a significant increase in [3H]TdR incorporation by human PBL and by human cord blood lymphocytes. The magnitude of the proliferative response of PBL to TGP does not correlate with the donor's titer of IgE antibodies to TGP, as assayed by a wheal and flare response after an i.d. injection of TGP, neither does it correlate with the donor's smoking history. [3H]TdR uptake is not observed before day 5 of culture, and the response peaks between days 5 and 10 of culture. Analysis of the cellular basis for the proliferative response suggests that T cells are proliferating. Two-parameter analysis by flow cytometry shows that CD3+, CD4+, and CD8+ cells are in the S + G2 + M phases, but not Ig-bearing cells or monocytes. A significant increase in HLA-DR (Ia)-bearing cells is observed on cells in all of the cell cycle phases. This increase coincides with cells entering the S phase. No increase is observed in the expression of the IL-2-R as assayed by the anti-Tac antibody. TGP also stimulates human PBL to differentiate and to produce Ig of the IgM, IgG, and IgA isotypes, without stimulating a detectable B cell proliferative response. The proliferative response of PBL is clearly due to TGP and not to contamination with LPS, because by the limulus amebocyte assay the TGP preparation contains less than 2% LPS, which could not account for the stimulation observed.

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In this study, we have demonstrated that antibody secretion by hybridoma cell lines can be down-regulated by idiotype-specific immune spleen cells or by nylon wool nonadherent spleen cells. This suppression of antibody secretion can be abolished by treating the idiotype-specific immune spleen cells with anti-Thy 1.2 plus complement. The hybridoma we used for most of our experiments secretes IgM specific for the cross-reacting haptens 2,4,6-trinitrophenyl (TNP) and 2,4-dinitrophenyl (DNP). Suppression was achieved by direct coculture of hybridoma cells with immune cells from animals which were injected with affinity-purified hybridoma antibody-coupled syngeneic spleen cells. The suppressed and control cultures contained similar numbers of viable hybridoma cells, suggesting that a simple cytotoxic effect is not responsible. Idiotype specificity was established in experiments showing that two idiotype immune animals immunized with antibody from two different IgM anti-TNP hybridomas could suppress the hybridoma to which they were immunized but could not affect the other hybridoma. Immune spleen cells required 3-4 days of coculture with hybridoma cells before maximum suppression was achieved. The kinetics of the response suggest that the final effector suppressor cell is generated during the coculture period and that a second signal, perhaps a product of the hybridoma cells, may be required.

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The proliferation of murine T lymphocytes in response to syngeneic Ia bearing non-T cells (syngeneic mixed lymphocyte reaction, SMLR) has been shown to generate regulatory T cells in vitro. An in vivo regulatory role has therefore been proposed for the SMLR. To study this role more directly, we examined the effects of repeated iv injection of mice with activated syngeneic B cells. Three such weekly injections induced a suppression of the plaque forming cell response to a subsequent injection of trinitrophenylated keyhole limpet hemocyanin (TNP-KLH). The suppression was transient and could not be maintained by additional injections of activated syngeneic B cells. The suppression was transferable to syngeneic recipients with splenic lymphocytes. Continued weekly iv injections of LPS induced blasts, as well as weekly intraperitoneal injections, caused enhancement rather than inhibition of the response to iv injected TNP-KLH. The enhancement was prevented by injection of anti-L3T4. Spleen cells from mice which had received three iv injections of activated syngeneic cells suppressed an in vitro secondary response to TNP-KLH by normal immune spleen cells. The cells responsible for the immune suppression were Thy 1.2+. The results indicate that repeated exposure to activated B cells causes activation of suppressor pathways but does not bring about a chronic state of immune suppression.

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We have shown that the mitogenic response of human peripheral blood lymphocytes (PBL) to tobacco glycoprotein (TGP), a glycoprotein rich in rutin or rutinlike polyphenol moieties, which is isolated from cured tobacco leaves, does not decrease with age. In contrast, the proliferative response to lipopolysaccharide (LPS) tends to decline with age, and a significant decrease is observed in the mitogenic response to rutin-bovine serum albumin (R-BSA). LPS and R-BSA are similar in some aspects to TGP, the former in that TGP and LPS are both T-independent B-cell mitogens for mice and are both highly negatively charged, and the latter in that covalently bound polyphenol groups are present on both R-BSA and TGP. Although the kinetics of the mitogenic responses to these three mitogens are similar (T. Francus, R. F. Klein, L. Staiano-Coico, G. W. Siskind, and C. G. Becker, Effects of tobacco glycoprotein (TGP) on the immune system. II. TGP is a mitogen for human peripheral blood lymphocytes. Submitted for publication.), multiple regression analyses show no correlation in the mitogenic responses of PBL from young donors to TGP and LPS, to TGP and R-BSA, or to LPS and R-BSA. In contrast, there are significant correlations between the proliferative responses to these mitogens by PBL from old donors. The results suggest that only a small subpopulation of the cells which are stimulated by R-BSA and LPS are not altered with age, and these are most likely the cells that are stimulated by the three mitogens studied.

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The effect of age on the regeneration of the B cell population was studied by cell transfer methods, using the allotype-congenic mouse strains BALB/c (Igha) and C.B-17 (Ighb) as donors of old and young bone marrow (BM) and spleen cells, and C.AL-20 (Igho) as recipients. This design allowed us to identify the origin of the sIgD+ B cells present in the recipients. It was found that in a simple cell transfer, BM cells or spleen cells of aged donors could reconstitute the peripheral B cell population of irradiated, thymectomized recipients essentially as effectively as could BM or spleen cells from young donors. However, when BM cells from aged donors and from young donors were mixed and were used to reconstitute a single recipient, the cells from the aged donor were less efficient than were the cells from the young donor. We found that sIgD+ B cells of young donor origin predominated in the peripheral B cell population of the recipient at 3 to 6 wk after cell transfer. In the BM of the recipients, however, there was no difference in the incidence of sIgD+ B cells derived from the young and the old donors. When recipients were reconstituted with a mixture of spleen cells from old and young mice, the sIgD+ cells of young donor allotype showed a tendency to predominate in the peripheral B cell population, although this predominance was not statistically significant. Under such competitive conditions, the spleen cells of aged donors were less efficient than the BM of aged donors in reconstituting the sIgD+ B cell population of the recipient's BM, but were more efficient in reconstituting the splenic sIgD+ cells. Thus, a subtle defect in the B cell precursor population of the BM and the spleen of aged mice has been demonstrated. The role of T cells in the generation of sIgD+ cells was also analyzed.

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It has been shown that tobacco glycoprotein (TGP), a polyphenol-rich glycoprotein antigen purified from cured tobacco leaves, is mitogenic for lymphoid cells in the spleen, peripheral blood, and bone marrow, but not for thymus cells. The proliferative response is not reduced by treatment of spleen cells or peripheral blood lymphocytes with anti-Thy-1.2 and complement, and spleen cells from the congenitally athymic (nu/nu) CD-1 proliferate as vigorously in response to TGP as do spleen cells from their heterozygous nu/+ littermates. In addition, TGP induces differentiation of mouse spleen cells into antibody-secreting cells, the majority of which secrete IgM, and the remainder mainly IgG and a few IgA. The differentiation into antibody-secreting cells induced by TGP occurs with spleen cells from nu/nu mice. It is concluded that TGP is a T-independent B cell mitogen for mouse lymphoid cells. On the basis of the ability of spleen cells from the LPS-nonresponder C3H/HEJ mice to respond to TGP with proliferation and differentiation into antibody-secreting cells, it is concluded that the effects of TGP are distinct from those of LPS and cannot be due to contamination of the TGP preparation with LPS.

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Evidence was obtained, by direct binding assays (radioimmunoassays) and by inhibition of binding assays, that after immunization some of the antibody molecules produced are degenerate in that they bind not only the immunizing antigen, but also unrelated ligands. It can be concluded that the exquisite specificity of the immune response is not necessarily a property of any given antibody molecule but is, at least to some extent, due to the summation of specificities held in common by the population of antibody molecules produced during the response.

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Tobacco glycoprotein (TGP) is a glycoprotein containing rutin-like polyphenol groups that is purified from cured tobacco leaves and can be detected in condensates of tobacco smoke. One-third of normal humans have been shown to manifest immediate, IgE-mediated, wheal and flare reactions to an intradermal injection of TGP. Rutin-like moieties are also found in a wide variety of vegetable foods. The possible importance of sensitivity to TGP in the pathogenesis of the vascular and pulmonary complications of tobacco smoking has stimulated us to study the immune response of mice to TGP and the role of rutin groups in influencing isotype expression. A series of three intradermal injections of TGP elicits a long-lasting IgE antibody response in mice. However, no hemagglutinating antibodies are produced. Similarly, immunization with a rutin derivative of bovine serum albumin stimulates IgE antibodies to bovine serum albumin but little hemagglutinating antibodies. In contrast, mice injected in the same manner with bovine serum albumin produce both IgE and hemagglutinating antibodies. Thus, the rutin moiety is implicated as exerting a regulatory effect on isotype expression by suppressing the production of serum antibodies of isotypes other than IgE. The immunization procedure employed (which involves an initial injection of 100 micrograms of antigen in phosphate-buffered saline, followed, at monthly intervals, by two intradermal injections of 100 micrograms of antigen precipitated on alum) apparently fails to stimulate the normal "down-regulation" of the IgE response so that a persisting high-titered response is obtained.

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In a cell transfer system, using the allotype congenic pair C57BL/6 (Ihgb) and B.C-9 (Igha), we established that the antibody response to dinitrophenylated bovine gamma globulin measured by solid state radioimmunoassay was only of donor allotype, both in the primary and in the secondary responses and regardless of the age of the cell donor. The magnitude of the response by allotype congenic recipients was similar to that of syngeneic recipients when either B.C-9 or C57BL/6 were used as donors.

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The autologous mixed lymphocyte reaction (auto-MLR) measures the ability of non-T cells to stimulate autologous T cells to proliferate in tissue culture. The auto-MLR was studied in 11 patients with autoimmune thrombocytopenic purpura (ATP). Seven patients had decreased auto-MLR, which averaged 4440 +/- 3364 cpm (SEM) compared to 15,360 +/- 6905 cpm for simultaneously studied controls. The average of the ratios of cpm incorporated by patients/cpm incorporated by control subjects was 0.20 +/- 0.06 (p less than 0.01). Serum from all 7 patients with low auto-MLR decreased the auto-MLR of normal subjects by an average of 56% +/- 8.5 (p less than 0.001). Preliminary results indicate that the inhibitory effect was mediated by a component of the IgG immunoglobulin fraction of serum. Sera from normal persons and from ATP patients with normal or high auto-MLR did not affect the auto-MLR of normal subjects. It was further shown that non-T cells from 3 of 5 patients with decreased auto-MLR failed to stimulate allogeneic T cells normally. It is concluded that many patients with ATP have decreased auto-MLR apparently due to the presence of a serum blocking factor and, perhaps, a defective stimulatory capacity of non-T-cells.

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