Monocyte suppression of Fusobacterium nucleatum-induced human polyclonal B-lymphocyte activation.

Previous studies reported that Fusobacterium nucleatum induced polyclonal B-lymphocyte activation (PBA) as determined by immunoglobulin M production in cultures of human peripheral blood mononuclear cells. However, the PBA response was greatly enhanced when the cells were depleted of esterase-positive, adherent cells (i.e., monocytes). The purpose of this study was to confirm and further examine the suppression of F. nucleatum-induced PBA (F. nucleatum-PBA) by blood monocytes. For comparison, PBA induced by pokeweed mitogen (PWM-PBA), which is enhanced by monocytes, was assessed in some experiments. We found the removal of monocytes from unfractionated cells by (i) Sephadex G-10, (ii) anti-monocyte specific OM-1 monoclonal antibody plus complement, or (iii) L-leucine methyl ester, a compound which selectively kills lysosome-rich cells, resulted in a population of cells responsive to F. nucleatum-PBA and unresponsive to PWM-PBA. The addition of double adherence-purified monocytes (greater than 85% esterase-positive cells), particularly in concentrations of greater than 10%, to lymphocytes depleted of monocytes by G-10, OM-1, or L-leucine methyl ester treatments, suppressed F. nucleatum-PBA and enhanced PWM-PBA. Monocytes also suppressed a mixture of isolated T and B cells combined in a T/B cell ratio of 3:1, which is an optimal ratio for F. nucleatum-PBA. Allogeneic monocytes suppressed F. nucleatum-PBA, although at low numbers these cells were not as suppressive as autologous monocytes. Heating at 56 degrees C for 15 min, sonicating, or freeze-thawing the monocyte preparations resulted in an abrogation of monocyte-induced suppression of F. nucleatum-PBA. Kinetic studies in which fresh monocytes were added daily to lymphocytes stimulated with F. nucleatum or PWM showed that the monocytes must be added within the first 2 days of culture to suppress F. nucleatum-PBA or enhance PWM-PBA. Monocytes incubated with F. nucleatum for 48 h released into the culture medium a soluble factor that suppressed F. nucleatum-PBA. The results from this study demonstrate a potent mechanism by which the host might prevent exaggerated nonspecific immunoglobulin responses when exposed to PBA-inducing concentrations of F. nucleatum. On the other hand, the induction of suppressive monocytes (or monocyte-mediated suppressive factors) by interaction with F. nucleatum might result in the inhibition of host protective immune reactions.     

Effect of Immunomodulating Factors Present in Ascitic Fluids and Sera From Cancer Patients on the Responses of Cultured Mononuclear Cells From Normal Subjects

ABSTRACT: Ascitic fluids and sera from patients with malignant tumors were tested for their ability to modulate the mitogen-induced blastogenic responses of normal subjects' peripheral blood mononuclear cells in vitro. The addition of either ascitic fluid or serum to cultures of normal blood cells greatly enhanced the blastogenic response of cells to phytohemagglutinin P, and markedly depressed the responses to concanavalin A and succinyl-concanavalin A. The blastogenic response of the cells to pokeweed mitogen was unaffected by the addition of serum and depressed by the addition of ascitic fluid. Autologous normal serum also enhanced the response to phytohemagglutinin P but had no effect on the response to the other mitogens. These activities were concentration-dependent and heat-stable (56°C, 60 min) and could be detected even if the ascitic fluid or serum was added as late as the second day of culture. Cells that had been preincubated with serum or ascitic fluid and washed well before culturing with the mitogens responded in the same manner as cells cultured in the presence of serum or ascitic fluid. The mitogen-induced blastogenic responses of mononuclear cells were not affected by the addition of autologous cells that had been preincubated with either serum or ascitic fluid, washed, and treated with mitomycin C. Indomethacin (2×10^?7M) did not prevent the ascitic fluid-mediated depression of blastogenic responses of normal cells. The ascitic fluid and serum of these cancer patients appeared to contain a specific immunoinhibiting substance which exerted its effects by a direct action on the responding mononuclear cells and not by the induction of suppressor cells.     

Polyclonal activation of human peripheral blood B lymphocytes by Fusobacterium nucleatum.

The present study examined in vitro polyclonal human B-lymphocyte (B-cell) activation (PBA) by Fusobacterium nucleatum. Pokeweed mitogen, a well-studied PBA activator, was included in some experiments for comparison. PBA was determined by the total immunoglobulin A, G, and M concentrations in the culture supernatants as measured by micro-enzyme-linked immunosorbent assay. F. nucleatum, at concentrations between 1 and 10 micrograms/ml, stimulated optimal PBA in monocyte-depleted cultures, whereas the pokeweed mitogen response was optimal in unfractionated, monocyte-containing cultures. Immunoglobulin synthesis occurred primarily between days 6 and 8 after stimulation with F. nucleatum. T lymphocytes enhanced the PBA response to F. nucleatum, particularly at a T- to B-cell ratio of 1:1. Immunoglobulin production was greater in round-bottomed wells than in flat-bottomed wells at lymphocyte concentrations of 200,000 cells per well. The PBA response, however, increased dramatically in flat-bottomed wells containing higher lymphocyte concentrations, suggesting that PBA is enhanced by cell-to-cell contact. A delay in stimulation of the lymphocytes with F. nucleatum resulted in diminished immunoglobulin production. The results provide information on the regulation of in vitro PBA induced by F. nucleatum. The data also suggest that there may be differences in the mechanisms by which F. nucleatum and pokeweed mitogen stimulate PBA.     

The maintenance of B-cell and T-cell function in frozen and stored human lymphocytes

The objective of this study was to identify and test a convenient means for long-term storage of lymphocytes taken from clinically characterized patients without losing B- or T-cell function. Accordingly, peripheral blood lymphocytes were frozen and stored, and portions of each sample were subsequently assayed for T-cell blastogenic response and B-cell Jerne plaquing at various time intervals after freezing. A comparison of the cell counts of fresh and frozen cultures indicated that all cells were recovered after freezing. Furthermore, these cells showed no significant differences in (i) cell viability; (ii) blastogenic response to antigens ofActinomyces naeslundii, Bacteroides melaninogenicus, Fusobacterium nucleatum, and tetanus toxoid; (iii) blastogenic response to phytohemagglutinin and pokeweed mitogen; and (iv) polyclonal B-cell response to pokeweed mitogen as measured by the direct Jerne plaque assay. The retained blastogenic and plaquing responses seen in frozen cultures indicated the maintenance of both T-cell and B-cell function, respectively. This is the first reported demonstration of Jerne plaquing of normal human lymphocytes after freezing. It appears that freezing techniques provide a means for repeating and extending both T- and B-cell assays using frozen stored portions of the same cell sample.     

抗人CD134单抗对外周血单个核细胞穿孔素mRNA表达的影响

目的： 观察不同浓度抗人CD134单抗对外周血单个核细胞穿孔素mRNA表达的影响及时间效应，旨在探讨抗人CD134单抗延长T细胞存活的可能机制。 方法: 应用反转录-聚合酶链反应(RT-PCR)技术分别检测不同浓度（1 mg/L、5 mg/L、10 mg/L）抗人CD134单抗对外周血单个核细胞穿孔素mRNA表达的影响，同时检测每种浓度作用不同时间（6 h、12 h、24 h、48 h）对外周血单个核细胞穿孔素mRNA表达的影响。 结果: 不同浓度CD134单抗作用不同时间，对人外周血单个核细胞穿孔素mRNA表达均有不同程度抑制作用，在 24 h 该作用达高峰。当CD134单抗浓度﹥5 mg/L时，这种抑制作用不再增强（P>0.05）。 结论: 抗人CD134单抗可在转录水平抑制穿孔素表达，该效应在 24 h 达高峰且会饱和，此乃抗人CD134单抗延长T细胞存活的主要机制。     

Induction of apoptotic cell death in peripheral blood mononuclear and polymorphonuclear cells by an oral bacterium, Fusobacterium nucleatum

It is largely unknown why a variety of bacteria present in the oral cavity are capable of establishing themselves in the periodontal pockets of nonimmunocompromised individuals in the presence of competent immune effector cells. In this paper we present evidence for the immunosuppressive role of Fusobacterium nucleatum, a gram-negative oral bacterium which plays an important role in the generation of periodontal disease. Our studies indicate that the immunosuppressive role of F. nucleatum is largely due to the ability of this organism to induce apoptotic cell death in peripheral blood mononuclear cells (PBMCs) and in polymorphonuclear cells (PMNs). F. nucleatum treatment induced apoptosis of PBMCs and PMNs as assessed by an increase in subdiploid DNA content determined by DNA fragmentation and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling assays. The ability of F. nucleatum to induce apoptosis was abolished by either heat treatment or proteinase digestion but was retained after formaldehyde treatment, suggesting that a heat-labile surface protein component is responsible for bacterium-mediated cell apoptosis. The data also indicated that F. nucleatum-induced cell apoptosis requires activation of caspases and is protected by NF-kappaB. Possible mechanisms of F. nucleatum's role in the pathogenesis of periodontal disease are discussed.     

Temporal cytokine profiling of Francisella tularensis-infected human peripheral blood mononuclear cells

BACKGROUND AND PURPOSE: Francisella tularensis is an intracellular bacterium known to replicate in monocytes and macrophages and cause tularemia in humans. Because of its infectious nature, F. tularensis is considered a biowarfare agent. Early cytokine profiles of Francisella-infected human peripheral blood mononuclear cells were evaluated. METHODS: Populations of human peripheral blood mononuclear cells were infected in vitro with F. tularensis live vaccine strain at a very low multiplicity of infection of 1:10 (bacteria:cells). A multiplex bead kit which analyzes 30 cytokines, chemokines and growth factors was utilized to measure secreted cytokines in cell supernatants 1, 4, 8, 16, and 24 h post-infection. RESULTS: Compared with uninfected controls, infected cells showed no increase in cytokine secretion at 1 and 4 h, implying a threshold for activation of immune responses. Starting at 8 h post-infection and continuing through to 24 h, an array of cytokines and growth factors was secreted by the infected cells. Some cytokines not previously associated with Francisella infection in humans were detected at 8 h, including interleukin-17 and interleukin-1 receptor agonist and vascular endothelial growth factor. CONCLUSIONS: The cytokine profiles of F. tularensis-infected peripheral blood mononuclear cells indicate an intricate pattern of both pro- and anti-inflammatory responses, including early T-cell activation.     

The effect of bacterial lipopolysaccharide (LPS) on histamine release from human basophils. I. Enhancement of immunologic release by LPS

Preincubation of human basophils with bacterial lipopolysaccharide (LPS) purified from the heptose-deficient mutant Salmonella minnesota R595 enhanced by an average of sixfold the response of peripheral blood basophils obtained from allergic donors to several allergens in vitro as judged by release of histamine. Enhancement occurred at suboptimal, optimal, and supraoptimal concentrations of antigen. No effect was seen if basophils were from a nonallergic donor, and LPS by itself rarely caused histamine release from any preparation of basophils. However, histamine release in basophils from nonallergic donors induced by antibody directed against IgE (anti-IgE) also was enhanced by LPS. Potentiation of histamine release occurred if basophils were pretreated with LPS before addition of anti-IgE for as little as 5 min; there was no increase in release if anti-IgE and LPS were added simultaneously to cells. LPS enhanced the rate of release without altering duration of the release response. LPS potentiation of release of histamine by F(ab')2 fragments of anti-IgE was equivalent to its effect on release triggered by the intact antibody molecule, confirming that the effect of LPS is not due solely to its interaction with the Fc component of the anti-IgE. These data thus provide evidence for modulation of basophil response to IgE-mediated stimuli by LPS, resulting in a significant enhancement of response. Enhancement by LPS appears to be independent of the stimulus which triggers the IgE receptor. The contribution of this mechanism to allergic disease or asthma remains to be determined.     

Formosanin-C, an immunomodulator with antitumor activity

Paris formosana Hayata (Liliaceae) grown in the mountain areas of Taiwan, has been used as a folk remedy for snake bite, and as an anti-inflammatory or anti-neoplastic agent. The effects of formosanin-C, a diosgenin saponin isolated from Paris formosana, on immune responses and transplantable murine tumor were studied. In culture systems, formosanin-C (0.03-0.16 microM) displayed significant enhancement of the blastogenic response of human peripheral blood cells to phytohemagglutinin. Formosanin-C also significantly increased the 3H-thymidine incorporation of ConA-stimulated lymphocytes at concentrations of 0.1 and 0.01 microM. The responsiveness of the granulocyte/macrophage colony-forming cells (GM-CFC) to mouse fibroblast cells L929 conditioned medium was altered in the presence of 0.01 and 0.001 microM of formosanin-C. In addition, formosanin-C given intraperitoneally activated natural killer cell activity at doses of 1-2.5 mg/kg. An intraperitoneal injection of 2.5 mg/kg of formosanin-C markedly induced interferon production, the peak blood level of which was observed 24 h after formosanin-C injection. Growth of subcutaneously transplanted MH134 mouse hepatoma was retarded by intraperitoneal treatment with 1-2.5 mg/kg of formosanin-C. The activity of 5-fluorouracil against MH-134 mouse hepatoma was potentiated by intraperitoneal treatment with formosanin-C. These results suggest that formosanin-C might display antitumor activity in association with modification of the immune system.     

Lectinlike interactions of Fusobacterium nucleatum with human neutrophils.

Fusobacterium nucleatum expresses lectinlike adherence factors which mediate binding to a variety of human tissue cells. Adherence is selectively inhibited by galactose, lactose, and N-acetyl-D-galactosamine. In this study, adherence of F. nucleatum to human peripheral blood polymorphonuclear neutrophils (PMNs) was investigated. The results indicated that the fusobacteria adhered to live and metabolically inactivated or fixed PMNs. Adherence of F. nucleatum resulted in activation of PMNs as determined by PMN aggregation, membrane depolarization, increased intracellular free Ca2+, superoxide anion production, and lysozyme release. Transmission electron micrographs showed that F. nucleatum was phagocytized by the PMNs. Microbicidal assays indicated that greater than 98% of F. nucleatum organisms were killed by PMNs within 60 min. Adherence to and activation of PMNs by F. nucleatum were inhibited by N-acetyl-D-galactosamine or lactose greater than galactose, whereas equal concentrations of glucose, N-acetyl-D-glucosamine, mannose, and fucose had little or no effect on F. nucleatum-PMN interactions. Pretreatment of the fusobacteria with heat (80 degrees C, 20 min) or proteases inhibited adherence to and activation of PMNs, but superoxide production was also stimulated by heated bacteria. The results indicate that interaction of F. nucleatum with PMNs is lectinlike and is probably mediated by fusobacterial proteins which bind to other human tissue cells. Adherence of F. nucleatum to PMNs in the absence of serum opsonins, such as antibodies and complement, may play an important role in PMN recognition and killing of F. nucleatum in the gingival sulcus and in the subsequent release of PMN factors associated with tissue destruction.     

Conditions for the enhancing effect of protease inhibitors on the concanavalin A induced thymidine response of murine lymphocytes

Incorporation of [3H]-thymidine - [3H]-TdR - into concanavalin A (Con A) stimulated murine splenocytes and thymocytes was found to be enhanced by addition of certain concentrations of phenyl-methylsulfonylfluoride (PMSF), di-isopropylfluorophosphate (DFP), N-alpha-tosyl-L-lysyl-L-chloromethylketone (TLCK), and soybean trypsin inhibitor (SBTI). No enhancement could be observed when mononuclear cells of the peripheral blood were used, and a medium enhancement when thymocytes were applied. Furthermore, no enhancing effect of the protease inhibitors (PI) on the Con A response of murine splenocytes could be observed within the first 24 h of the culturing period. DFP, PMSF, and TLCK enhanced the Con A response to a similar degree, whereas SBTI was less effective. DFP and SBTI proved to be also effective when they were added after 15-24 h to the Con A cultures, if the cultures were harvested 48 h later. Removal of adherent and phagocytic spleen cells or reduction of the concentration of spleen cells shifted the effective DFP concentration to lower concentrations, whereas addition of adherent spleen cells caused a shift of the enhancing DFP amounts to higher concentrations. The data presented suggest that the enhancing effect of PI on the T cell response depends on the concentration of PI, the time of culturing and incubation, the PI used, the origin of the stimulated cells, and especially on the number of adherent and phagocytic cells. These findings might explain - at least in part - the different results on the effect of PI on the T cell response obtained in the past.     

Changes in leukocyte recirculation, NK cell activity, and HLA-DR expression in peripheral blood mononuclear cells of MS patients treated with Poly ICLC.

To investigate the cellular immune effects of the interferon inducer, Poly ICLC, in humans, peripheral blood mononuclear cells from patients with multiple sclerosis receiving Poly ICLC as part of a preliminary clinical trial were studied. Peripheral blood mononuclear cell phenotype analysis using fluoresceinated monoclonal antibodies and flow microfluorometry showed decreases in the percentages and absolute numbers of all lymphocyte subsets 24 h after infusion. These changes returned toward baseline at 48 h except the percentage of CD-4 positive cell which increased above baseline levels. The percentage of HLA-DR antigen positive cells and CD-16 (Leu 11a) positive cells were increased 24 h after infusion but returned to baseline at 48 h. NK activity as determined by chromium release from K562 target cells was decreased at 24 h but increased 48 h after drug infusion. The increases in percentages of HLA-DR antigen and CD-16 positive cells at 24 h and NK activity at 48 h are consistent with the in vitro effects of IFN while the decreases in peripheral blood mononuclear cells are suggestive of changes in cell recirculation.     

Effect of cyclosporin A on in vitro proliferative activity and immunoglobulin synthesis of isolated human lymphoid cell subpopulations.

Cyclosporin A inhibited at equal concentration both pokeweed mitogen and Staphylococcus aureus Cowan I-induced in vitro proliferative activity of blood leucocytes, intracellular immunoglobulin synthesis and release of immunoglobulin to the culture medium. When the interacting lymphoid cell subpopulations, T cells, B cells, T gamma and T micro cells were fractionated apart and separately stimulated with these mitogens, cyclosporin A inhibited again at equal concentrations pokeweed mitogen-induced proliferation of blood T gamma, T micro and B cells and Staphylococcus-induced proliferation of B cells. The 50% inhibitory concentration varied in separate experiments between 10(-2)-10(-1) microgram/ml. Although cyclosporin A efficiently suppressed the blastogenic response, a 100- to 1,000-fold concentration of the drug was required to damage resting lymphocytes in culture. After the blastogenic phase, pokeweed mitogen-stimulated blood leucocytes were again resistant to cyclosporin A: intracellular Ig synthesis and release of Ig to the culture medium proceeded independently of the presence of the drug. As the phagocytic activity of mononuclear phagocytes was not affected by prior culture in cyclosporin A-containing medium, we conclude that in man cyclosporin A suppression both T and B lymphocyte blastogenesis, and that the suppression is due to a direct effect on the blast cell rather than mediated via a third-party accessory cell.     

Sensitivity of preincubated lymphocytes to suppressor regulation

DNA synthesis of human peripheral blood lymphocytes increases if Con A is added to the culture after 24 h preincubation at 37 degrees C. During preincubation the mitogen reactive lymphocytes lose their sensitivity to the suppressive effect of autologous mitomycin-treated mononuclear leukocytes, and of supernatants of autologous preincubated cells. The reactive lymphocytes preincubated at 4 degrees C retain their sensitivity to the suppressive effect of regulatory cells and their supernatants. It is assumed that the enhancement of mitogen response after preincubation at 37 degrees C is caused by a decrease of suppressor regulation of human lymphocytes. Prostaglandins may be regarded as one of the mediators of the suppression.     

Mechanism of immunosuppression in leprosy: presence of suppressor factor(s) from macrophages of lepromatous patients.

Human peripheral blood mononuclear cell proliferation induced by Mycobacterium leprae could be inhibited by the suppressor factor in the lysate of the macrophages of lepromatous leprosy patients. Macrophages from normal subjects and tuberculoid patients did not show production of a suppressor factor. Inhibition occurred only when the factor was present in the initial stages of lymphocyte culture. The factor is heat stable and nondialyzable. Proliferation induced by some mycobacteria and concanavalin A could also be blocked by the factor. Interestingly, blastogenic response by a few other antigens and phytohemagglutinin could not be inhibited by the suppressor factor. Mononuclear cells pretreated with such lysate from lepromatous macrophages for 24 h could induce suppressive activity in the cells in vitro in an autologous system. Treatment of these cells with carbonyl iron after the induction phase, to remove phagocytic cells, did not abolish their suppressive activity. The lepromatous macrophage lysate also generated suppressive activity in a T-lymphocyte-enriched population of normal subjects. These studies are interpreted to indicate that immunosuppression in lepromatous patients is produced by both macrophages and T lymphocytes. The exact phase in which either of these cells acts as a suppressor may be different. Specific suppression by macrophages to M. leprae can be an early event, and nonspecific suppression by T lymphocytes may be a later event in the course of lepromatous leprosy.     

IL-2 receptor gene expression and IL-2 production by human preterm newborns' cells.

IL-2 receptor (IL-2R) gene expression in human umbilical cord blood mononuclear cells (CBMC) of preterm and term newborns was examined following stimulation for 18 h with phytohaemagglutinin (PHA) and compared with that of adult peripheral blood mononuclear cells (PBMC; mothers and control group). mRNA for IL-2R could not be detected in CBMC of preterm infants, whereas the mRNA levels for IL-2R found in full term neonates were similar to those observed in PBMC of adults. IL-2 activity in conditioned medium (CM) of mononuclear cells stimulated with either optimal or suboptimal PHA concentrations for 24 h and 48 h was also determined. At 24 h of stimulation, IL-2 activity found in CM obtained from CBMC of preterm and term newborns was significantly higher than that found in CM of adults' PBMC. A further enhancement of IL-2 activity (six to eight times) was observed in CM of preterm and term cells stimulated for 48 h, whereas no significant difference was found in IL-2 activity in CM from adult cells tested at the two incubation periods. The present findings may provide an additional explanation for the impaired function of the immune system, and the high susceptibility to infections observed in preterm newborns.     

Blastogenic responses to Pneumocystis carinii among patients with human immunodeficiency (HIV) infection.

Proliferative responses by human peripheral blood mononuclear cells and rat spleen cells were measured to Pneumocystis carinii in a blastogenic assay using organisms obtained from rat lungs and propagated in tissue culture as the antigen. Responses occurred only in subjects with known prior exposure to P. carinii and the magnitude of the response varied with the number of organisms present in the antigen preparation and days in culture. Healthy human adults showed higher proliferative responses to P. carinii than did patients with Class II (asymptomatic) or Class III (lymphadenopathy) human immunodeficiency virus (HIV) infection. No responses to the preparation were found among Class IV HIV patients with the acquired immunodeficiency syndrome, including those with prior episodes of P. carinii pneumonia or those receiving azidothymidine. Overall, the blastogenic responses obtained with P. carinii were similar to those obtained with tetanus toxoid and phytohemagglutinin, and correlated well with the number of circulating CD4 cells. The data suggest that the blastogenic assay using tissue culture-derived rat P. carinii is specific for the organism and should be helpful in studying the cellular immune responses in pneumocystosis among different human patient populations.     

Modulation of immunoreactivity to periodontal disease-associated microorganisms during pregnancy.

The lymphocyte blastogenic response to a panel of antigens and mitogens was assessed in a group of 20 women throughout their pregnancy. In addition, a group of five nonpregnant women was monitored simultaneously to identify variations in response to the same stimulants. The stimulants included orally associated bacterial antigens (Streptococcus sanguis, Actinomyces viscosus, Bacteroides asaccharolyticus, Bacteroides melaninogenicus subsp. intermedius, Bacteroides [Capnocytophaga] ochraceus, and Fusobacterium nucleatum) and non-orally associated-stimulants (streptokinase-streptodornase, tetanus toxoid, concanavalin A, phytohemagglutinin, and pokeweed mitogen). Intrinsic (cells cultured in male AB plasma) suppression of the lymphocyte response to these stimulants was observed to occur by the second trmester of pregnancy and was resolved after parturition. Additionally, an extrinsic (cells cultured in autologous plasma) suppression was also suggested to occur in a similar manner. There was no detectable enhancement of the blastogenic response to oral bacteria associated with elevated gingivitis, which is generally reported to occur during nonpregnancy gingivitis. We propose that concomitant immunosuppression occurs during the second trimester, which masks such enhancement.     

Induction of suppressor cells in normal lymphocytes by uremic serum

Sera of patients on chronic hemodialysis induced suppressor cell activity (SCA) in normal peripheral blood mononuclear cells, which significantly impaired blastogenic response to PHA. This SCA is statistically not different from Con A induced SCA. Both SCAs are however additive. Speculations concerning the modes of action of this induced SCA are discussed.     

Mitogen-induced amplification of blastogenesis in lipopolysaccharide-precultured lymphocytes.

Human peripheral blood lymphocytes were cultured in vitro with lipopolysaccharide (LPS) for 48 h. After washing, stimulation with a concanavalin A (ConA) or pokeweed mitogen (PWM) resulted in a synergistic blastogenic response that was greater than the sum of the independently stimulated control cultures. Addition of fresh autologous lymphocytes after LPS preculture produced an additional increment of synergy. The nature of the responding and helping effects was determined by coculturing irradiated or nonirradiated lymphocyte suspensions that had been precultured with either LPS or ConA/PWM. Such studies indicated that amplification was the result of a mitogen-activated helper activity, which facilitated the blastogenic response to LPS. Experiments with lymphocytes resolved into T- and B-cell-enriched fractions indicated that the LPS-responsive cells were of the B type and that the help was provided by a mitogen-activated T-cell population. These studies indicated that LPS can induce human B-cell blastogenesis; however, a helper function must be provided by a T-cell subpopulation. This helper activity is inducible by pretreatment of the T cells with plant lectins.