Polyclonal activation of B lymphocytes by lipopolysaccharide requires macrophage-derived interleukin-1.

Lipopolysaccharide (LPS) is a potent murine polyclonal B-cell activator which induces cellular proliferation and IgM secretion. The precise role of activated macrophages in the induction of LPS-dependent, B-cell responses has been unclear. Although early reports concluded that the LPS effect occurs independently of other cell types, other studies have suggested that adherent macrophages exert either potentiating or inhibitory effects. In the present study, B-cell mitogenesis and IgM production were measured in primary spleen cell cultures after removing adherent cells by a variety of experimental procedures. B-cell activation by LPS was found to be strictly dependent on the presence of adherent macrophages. Antibody neutralization and cytokine reconstitution studies demonstrated that macrophage-derived interleukin- (IL-1) is a necessary co-factor for LPS-induced polyclonal activation.     

Regulation of T- and B lymphocyte responses to mitogens by tumor-associated macrophages: the dependency on the stage of tumor growth

This preliminary investigation was concerned with the hypothesis that macrophages associated with tumors induced to regress temporarily by the action of cyclophosphamide (CY) have the capacity to suppress local T- or B lymphocyte responses which otherwise might cause permanent regression. Cultures of adherent cells, predominantly tumor-associated macrophages ( TAMs ), isolated from two C57BL/6J (B6) sarcomas, MCA/76-9 and 76-64, after various periods of tumor growth or after CY injection (240 mg/kg) were shown to suppress or enhance the response of 10(6) B6 normal spleen cells to stimulation by concanavalin-A (Con A) or lipopolysaccharide (LPS). At any given time point, the extent of suppression or enhancement induced by adherent cells isolated from tumors after CY injection was similar to that induced by cells from progressing tumors and appeared to be more dependent on the time after the initial injection of tumor cells than on drug treatment per se. Thus, adherent cells suppressed Con A and LPS responses when they were isolated either from small (0.5 g) or large (greater than 1.5 g) progressing tumors or from tumors 4-9 days after CY injection. However, during the logarithmic phase of tumor growth or within 4 days of injecting CY, adherent cells enhanced spleen cell mitogenic responses, particularly to LPS stimulation. The cultures of putative TAMs isolated at the various time points were seen to contain varying proportions of polymorphonuclear cells (PMNs), the proportions coinciding with the period of reduced spleen cell mitogenic responses. Cultures prepared from small or large progressing tumors contained about 10% or 20-30% PMNs, respectively, while those from tumors tested during the log phase contained less than 5% PMNs. Cultures from tumors removed within 4 days of injecting CY contained less than 5% PMNs while those prepared from tumors tested at later times contained as high as 25% PMNs by 9 days after CY injection. A comparison of peritoneal PMNs and macrophages from non-tumor-bearing mice in terms of their ability to influence mitogenesis indicated that PMNs at high ratios (1 PMN:2 spleen cells) could suppress responses directly. Lower ratios had either small suppressive effects or no effects but in no case was enhancement of responses seen. Peritoneal macrophages suppressed responses at a ratio of 1 macrophage:10 spleen cells but enhanced responses at ratios of 1 macrophage:100 or 1,000 spleen cells. The overall data indicated that the microenvironment of the tumor site may modulate the functional activity of macrophages, or their subpopulations, during their potential regulation of T- and B-cell responses.(ABSTRACT TRUNCATED AT 400 WORDS)     

Suppression of Immunological Activities by Mouse Amniotic Fluid

Mouse amniotic fluid (MAF) was shown to be capable of suppressing those antibody responses observed in euthymic or athymic mouse spleen cell cultures to the T-independent antigens dinitrophenylated Ficoll (DNP-Ficoll) and trinitrophenylated lipopolysaccharide (TNP-LPS) and to the polyclonal B-cell activators LPS and purified protein derivative of tuberculin (PPD). Titration experiments demonstrated that the suppressive capacity of MAF for either LPS or DNP-Ficoll responses was maintained up to a MAF dilution of 1:120. Preincubation of spleen cells obtained from athymic mice with MAF for 8 h significantly suppressed polyclonal B-cell activation of such cells induced by LPS, although suppression was greater when MAF was present during the entire culture period. In addition, the suppressive activity that MAF demonstrated for antibody production induced by DNP-Ficoll or LPS was not lost as a result of dialysis. MAF also suppressed the secondary in vitro proliferative responses of lymph node cells sensitized to the T-dependent antigen human gamma globulin (HGG). HGG-induced proliferation of such cells appeared to be more susceptible to suppression effected by MAF than concanavalin-A-induced proliferation.     

Analysis of suppressor factor in delayed immune responses of a bat, Pteropus giganteus

Bat spleen and mesenteric lymph node cell cultures treated with varying doses of LPS showed significant blastogenic and DNA synthetic responses between 72-96h. Peak responses were not different when the cell culture medium was supplemented either with autologous serum or heterologous serum indicating the absence of any significant suppressor factor in autologous bat serum. In contrast, blastogenesis and DNA synthesis peaks appeared early, at 48h, in lymphocytes depleted of suppressor T cells by pretreatment with cyclophosphamide. Direct antibody producing cells against SRBC were studied in normal spleen and mesenteric lymph node cell cultures. The peak PFC response was lacking even at 120h while CY pretreated bat lymphocytes, showed peak PFC responses at 96h. Thus the delayed immune response in bats seems to be a function of suppressor T cells but serum suppressor factor(s) possibly exerts no significant effect. The function of suppressor cells in bats in relation to their role as carriers of several dreaded bacteria and viruses is discussed.     

Alveolar macrophages. II. Inhibition of lymphocyte proliferation by purified macrophages from rat lung.

Macrophages were prepared from the lung, peritoneal cavity and blood of normal, unstimulated rats from a number of strains. The macrophages were purified by adherence, and characterized via surface markers, enzyme activity and phagocytic capacity, and subsequently tested for activity in cultures of mitogen-stimulated syngeneic lymphocytes. Peritoneal macrophages and blood monocytes were mildly stimulatory, or ineffective in modulating mitogen-induced DNA synthesis; peritoneal macrophages reconstituted the blastogenic responses of macrophage-depleted lymph node cell cultures to normal limits. In contrast, alveolar macrophages were markedly inhibitory to lymphocyte proliferation; in some instances inhibitory activity was demonstrable when added alveolar macrophages comprised only 0.04% of the total cells in culture. Lymphocyte proliferation induced by T-cell mitogens was more susceptible to this inhibition than was proliferation induced by the B-cell mitogen LPS. Alveolar macrophages recovered from SPF rats, while less in number, exhibited comparable inhibitory activity. These results form part of an emerging picture picture of the normal alveolar macrophage as a potential 'suppressor' of T-cell activity in the lung.     

Studies on antibody induction in vitro: II. Cellular requirements for a primary response to soluble T2 antigens by rabbit cell cultures

The primary in vitro response of cultured rabbit spleen cells to solubilized T2 phage (S-T2) was used to study the cellular requirements for immune in-induction. Rabbit lymph node cells, in contrast to spleen cells, were found not to respond to S-T2, and the addition of spleen cells insufficient in number to give rise to antibody in themselves failed to supply a missing cell type to lymph node cell cultures. The period in culture during which antibody formation by spleen cells could be induced was shown to be transitory, in that addition of S-T2 even 24 hours after culture initiation resulted in little, if any, antibody production. Cultures of non-adherent cells from spleen, obtained by removal of adherent cells on plastic dishes or glass bead columns, by silica treatment for removal of phagocytic cells, or by sequential treatment by adherence and with silica, responded as well to S-T2 as did the whole spleen cell population. These findings strongly indicate the relative macrophage independence of the in vitro response to S-T2 and are suggestive of the role of macrophages in solubilization of antigens, since intact phage fails to induce antibody formation in macrophage-deprived cultures.     

DNA synthesis and production of interleukin 1 by lymph node macrophages in culture

When bovine lymph node cells are cultured for several days the adherent macrophage population increases by as much as tenfold. This increase in cell number is primarily due to cell division, which reaches a maximum on day 4 or 5 of culture. Although the presence of the nonadherent cells seems required for cell division, we have been unable to detect a macrophage growth factor in either the nonadherent cell populations. The adherent cells were identified as macrophages based on positive esterase staining, the presence of Fc receptors, beta-glucuronidase activity, and phagocytosis. Moreover, these adherent cells produced interleukin 1 (IL1) after exposure to lipopolysaccharide in serum-free medium. Approximately 10(7) macrophages were stimulated to produce about 900 units of IL1 in a 24-hr period. Thus, the bovine lymph node preparation is a potential source of a large number of macrophages capable of dividing in culture and of producing IL1.     

Lymphocyte function in experimental endemic syphilis of Syrian hamsters.

We have studied the changes in the lymph nodes, spleen and thymus that occur in inbred LSH Syrian hamsters infected with Treponema pallidum Bosnia A, the causative agent of endemic syphilis, as well as the B-cell responses of these infected animals to helper T-cell independent and dependent antigens. The lymph nodes increased significantly in weight up to 6 weeks after infection, and contained viable treponemes. No significant changes in the spleen weight were observed, and no viable treponemes could be recovered from the spleen. However, the size of the thymus decreased steadily during the course of the disease. The relative number of Ig+ cells (B cells) increased in the spleen and regional lymph nodes, whereas the relative number of T cells decreased during the course of infection. In both the spleen and lymph nodes, the relative number of macrophages increased initially and decreased thereafter in the form of a bell-shaped curve showing a peak at 4-6 weeks of infection. The ability of splenic lymphocytes from infected hamsters to mount a primary PFC response to pneumococcal polysaccharide type III (SIII), a helper T-cell independent antigen, was elevated throughout the course of infection. However, the splenic PFC response to sheep erythrocytes (SRBC), a helper T-cell dependent antigen, was increased only during the first 4 weeks of infection and progressively decreased thereafter. The PFC responses of infected lymph node lymphocytes to both SIII and SRBC were increased during the first 4 weeks and decreased thereafter. These data suggested that atrophy of the thymus seen in syphilitic infection is accompanied by the complex losses of subsets of T cells and altered B-cell functions. An early loss of suppressor T cells in both the lymph nodes and spleen occurs concomitantly with a loss of T helper cells and heterologous (treponema-unrelated) B-cell functions in the lymph nodes. Helper T cells are lost from the spleen only in the later stages of infection, whereas splenic B-cell functions remain intact throughout the course of the disease. These findings were further tested by in vitro methods where splenic and lymph node lymphocytes from infected hamsters were examined for their ability to respond to Con A in terms of the induction of antigen non-specific suppressor T cells. The mixing of Con A stimulated splenic or lymph node lymphocytes from infected hamsters was unable to inhibit the primary antibody responses of SRBC as compared to the normal control.(ABSTRACT TRUNCATED AT 400 WORDS)     

Direct Activation of Guinea Pig Macrophages by a B-Cell Mitogen

Since polyclonal B-cell activators (PBA) require macrophages to induce modifications in the lymphocyte in vitro primary response to thymus-dependent antigens, we have investigated whether PBA act directly on macrophages. [14C]glucosamine uptake by guinea pig peritoneal adherent cells after stimulation with purified protein derivative (PPD), lipopolysaccharide (LPS), and dextran sulfate (DxS) was tested. PPD produced an increased [14C]glucosamine uptake, whereas LPS and DxS did not. According to our experiments, (a) PPD does not require the presence of lymphocytes to stimulate macrophages; furthermore, when lymphocytes were present in a concentration higher than 5%, a suppressor effect in the glucosamine uptake was found, and (b) there was no significant difference between the findings when peritoneal adherent cells were cultured in normal medium and in supernatant of lymphocyte cultures stimulated with PPD.     

In vitro selection of murine antigen-specific T-lymphocytes. I. Description of selection procedure

Enrichment of antigen-responsive murine T-lymphocytes was achieved by two in vitro procedures through the preferential adherence of the antigen-specific cells to the antigen-pulsed macrophages and their consequent multiplication. The first procedure involved the addition of column-purified T-enriched lymph node lymphocytes from immunized mice to a monolayer of antigen-pulsed adherent spleen cells from non-primed syngeneic donors. Lymphocytes which failed to adhere to the antigen-pulsed monolayer were removed after 4 h of incubation. The adherent cells were cultured for a week and the lymphocytes obtained after that period from the selection plate were highly responsive to the antigen for which they were selected and for a T-cell mitogen (Con A). On the other hand, these selected cells demonstrated little or no response to other antigens, to which the original donor of the lymphocytes was immune, and to a B-cell mitogen (LPS). The same preferential response to the selecting antigen and T-cell mitogen was obtained in lymphocytes enriched in the alternate procedures on ‘supernatant cultures’. The enriched population from ‘supernatant culture’ was derived from cells that did not adhere to the antigen-pulsed monolayers during 4 h of incubation. The non-adherent lymphocytes which still contained antigen-specific lymphocytes were transferred to a fresh monolayer of antigen-pulsed adherent spleen cells to be grown for a week in culture. The improvement in the response to the selecting antigen and the decreased reaction to other immunizing antigens show that the cells harvested from either ‘selected’ or ‘supernatant’ cultures were enriched for a given antigen — the selecting antigen. In most individual experiments the enrichment was better in the selected cultures.The enrichment procedures were dependent upon effective antigen presentation to the lymphocytes. Spleen cells from mineral oil-injected mice, which are the most effective antigen-presenting cells, formed the most efficient monolayers for the enrichment of the antigen-specific lymphocytes, both in the ‘selected’ and ‘supernatant’ cultures.     

Macrophage Requirement for the in Vitro Response to an Insolubilized T-independent Antigen

The role of macrophages in the in vitro response of mouse spleen cells to the insolubilized, T-independent antigen trinitrophenylated polyacrylamide (TNP-PAA) is demonstrated by the following points. The response is abolished by filtration on Sephadex G-10 and can be restored by the addition of splenic adherent cells, deficient in either B or T cells, or by 2-mercaptoethanol (2ME). It is suppressed upon elimination of phagocytic cells by silica, and restored by 2-ME. 2-ME can restore a normal response from zero, in cultures depleted of both adherent and phagocytic cells, and is efficient in the absence of mature T cells. Experiments in microcultures show that large numbers of macrophages can stimulate a supra-optimal response from B cells. This response is only obtained in the presence of the antigen, and is specific for TNP. These results show that microphages, probably by their polyclonal B-cell activator (PBA) property, play a role in the specific response to TNP-PAA. This prompts us to discuss the respective roles in the B-cell response of this PBA activity and of the interaction of the antigen with the specific B-cell receptors.     

Suppression of the polyclonal B-cell response to lipopolysaccharide by concanavalin-A-treated non-T cells in vitro.

Concanavalin A (Con A), a T-cell mitogen, dose-dependent suppressed the polyclonal antibody response of a purified B-cell population to lipopolysaccharide (LPS) in vitro. This suppression can be attributed to a role of suppressor cells generated in B-cell cultures in response to relatively high doses of Con A. The suppressor cells can be produced in cultures of normal as well as athymic nude spleen cells deprived of plastic-adherent cells and Thy 1.2 positive cells. The suppressing activity of Con-A-treated cells was not abolished by pre-treatment of the cells with anti-Thy 1.2, anti-Lyt 1.1, anti-Lyt 1.2 or anti-Lyt 2.2 serum and complement, and was decreased partially by treatment with anti-Ig serum and complement. Moreover, the suppressing activity was partially decreased by using petri dishes coated with IgG of F(ab')2 fraction of anti-mouse immunoglobulins. Thus, the suppressor activity of Con-A-treated cells was mediated by a non-T cell, possibly a B cell. A role for macrophages was unlikely, but not formally ruled out. The suppressor cells retarded development of polyclonal antibody production by B cells to LPS only when added at the start of culture and was resistant to treatment with anti-mitotic doses of irradiation and mitomycin C. It is possible that the suppressor cells of a B-cell nature play a role in the regulation of excessive B-cell proliferation during antibody responses.     

In vivo depletion of macrophages by desulfated iota-carrageenan in mice

Almost 90% of the sulfate groups of iota-carrageenan (CGN) was removed with acid-methanol in an attempt to obtain a product which would selectively eliminate macrophages in mice. Desulfated CGN(DS-CGN) failed to induce in vivo polyclonal antibody production in DBA/2 mice. However, the number of phagocytes in the peritoneal cavity, spleen, thymus and lymph node of DBA/2 mice was reduced stringently by DS-CGN treatment. The number of Mac-1 positive cells(macrophages) in DS-CGN-treated mice gradually decreased for at least 7 days after the last injection of DS-CGN. In contrast, the relative proportion of T and B lymphocytes in the lymphoid organs was unaffected by DS-CGN treatment. DS-CGN suppressed antibody responses to SRBC, a T cell and macrophage-dependent antigen, but no such suppressive effect was observed in the polyclonal antibody responses to LPS, a T cell and macrophage-independent B cell activator. Furthermore, the impaired SRBC antibody responses in DS-CGN-treated mice were restored following transfer of adherent cells but not T cells. These experimental results indicate that DS-CGN selectively eliminates macrophages without influencing lymphocyte function in vivo.     

Immunological competence in osteopetrotic (ia) rats

Osteopetrosis in ia rats is characterized by excessive skeletal mass and reduced bone resorption. The skeletal defects can be corrected by the transfer of mononuclear spleen cells from normal littermates. These studies suggest that osteopetrotic mutants may also have defective immune functions. The op, osteopetrotic, rat demonstrates early thymic atrophy and immune function which decreases with age. Several studies have shown significantly reduced responses to T and B cell mitogens by spleen cells from osteopetrotic mutant mice. The problem with these latter studies is that different populations of cells have been compared in mutants and normal littermates because the spleen is a focus of extramedullary hemopoiesis in osteopetrotic animals. To circumvent this problem, the Ficoll-Hypaque, mononuclear isolate of spleen and mesenteric lymph node from 5-week-old ia and normal littermates were compared. Under appropriate culture conditions the cells were exposed to Con A, PHA, and LPS for 3 days and 3H-thymidine for the last 24 hours. In all cases, the response to optimal concentrations of the 3 mitogens was similar for ia and normal spleen and lymph node cells (ia/control ratios ranged from 0.6 to 1.2). The cellular composition of the samples tested in the mitogen assays were also evaluated by fluorescent microscopy using FITC-conjugated monoclonal antibodies directed against specific cell surface markers. The percentage of B cells, macrophages, total T cells, and helper T cells were found to be similar in the Ficoll-Hypaque isolate of ia and normal spleen and lymph nodes. Likewise, the ia mutant does not show any signs of abnormal thymic involution. These results indicate normal immune function in the ia mutant when similar populations of cells are compared.     

Macrophage-Agglutinating Factor Produced In Vitro by BCG-Sensitized Lymphocytes

Supernatant fluids from cultures of BCG-sensitized rabbit lymph node and spleen cells contained a factor that strongly agglutinated normal rabbit alveolar macrophages within 3 min at room temperature. In contrast, fluids from nonsensitized cell cultures did not agglutinate normal rabbit alveolar macrophages. This factor was designated macrophage-agglutinating factor (MAgF) because it is similar to the previously described factor found in lung lavages of rabbits exhibiting a BCG-induced pulmonary delayed hypersensitivity reaction. The kinetics of MAgF production in vitro by sensitized lymph node cells and its inhibition by puromycin and actinomycin D suggest active synthesis; sensitized spleen cells exhibited kinetics resembling release rather than synthesis. Studies on purified lymphocyte and macrophage populations from sensitized spleen and lymph nodes indicated that lymphocytes are responsible for MAgF production. However, MAgF production was not induced in normal cells incubated in vitro with concanavalin A or phytohemagglutinin. Fractionation of cell culture supernatant fluids in Sephadex G-100 or Ultrogel AcA-34 clearly separated MAgF from migration inhibition factor; MAgF was present in the void volume of the eluates, suggesting a molecular weight of over 400,000, whereas migration inhibition factor was recovered in the same peak as albumin. The role of MAgF in vivo is unknown, but it is postulated that it may cause the adherence of macrophages during granuloma formation.     

Immunodepression in trypanosome-infected mice. VI. Comparison of immune responses of different lymphoid organs

Mitogen stimulation of cells from various lymphoid organs of C3H/He mice chronically infected with an isolate of Trypanosoma congolense was studied at different time intervals after infection, using concanavalin A (Con A) and lipopolysaccharide (LPS). At the same time, changes in the percentages of T, B and null lymphocytes in these organs were determined by immunofluorescence staining. The responses of T and B lymphocytes in the spleen were totally depressed, and the cellular composition was drastically altered by day 14 after infection. Unlike the spleen, the lymph nodes showed minor changes in their T and B lymphocyte responses and cell composition during the course of the infection, except the B cell response and composition which were altered late in the infection. The thymus and bone marrow did not show any appreciable changes in their mitogen responses and cell composition throughout the infection. The peripheral blood lymphocytes showed reduced B cell responses. Spleen cells from chronically infected mice suppressed lymphocyte stimulation induced in normal spleen and lymph node cell populations by Con A, LPS and allogeneic stimulator cells. Lymph node cells from the same group of mice did not exhibit any such suppressor activity. In the experimental system used here, the spleen is the primary site of immune depression, and other lymphoid organs such as the lymph nodes and thymus are very little affected.     

Suppression of the primary immune response to keyhole limpet haemocyanin by antimacrophage globulin.

Thioglycollate-stimulated, adherence and density gradient-enriched peritoneal exudate cells were used for the preparation of a specific, highly active antimacrophage reagent. After absorption with mouse lymphoid cells, an antimacrophage globulin (AMG) fraction was shown to be cytotoxic for peritoneal exudate macrophages but not spleen, lymph node or thymus cells. The AMG suppressed both the in vivo primary serum antibody and spleen PFC responses to KLH.     

Activation of Human Lymphocyte Subpopulations by Rabbit Anti-Human β2-Microglobulin and by Lipopolysaccharide

Rabbit anti-human beta2-microglobulin (anti-beta2m) was found to increase DNA synthesis in peripheral blood lymphocytes (PBLs) and in cells from abdominal lymph nodes, spleen, tonsil, adenoid, appendix, and bone marrow. The response to anti-beta2m was highest in cells originating from abdominal lymph node, appendix, and spleen. These organs were shown to contain a high proportion of surface-Ig-positive cells. No response to anti-beta2m was seen in thymus cells or in B-cell-depleted lymphocyte populations. Lipopolysaccharide (LPS) increased DNA synthesis in spleen cells, bone marrow cells, tonsil cells, and, sometimes, in cells from abdominal lymph nodes but weakly or not at all in PBLs. To study whether anti-beta2m and LPS activated the same subpopulation of lymphocytes, cultures were exposed to both mitogens in various concentrations. The effect on DNA synthesis in spleen cells was almost additive. This may indicate that these two polyclonal B-cell activators (PBAs) stimulate mainly distinct subsets of B cells in spleen. On the other hand, these two mitogens have a synergistic effect on DNA synthesis in PBLs. Since anti-beta2m is the first described selective B-cell mitogen activating human PBLs, it might be of clinical importance in the functional characterization of lymphocyte subpopulations.     

Immunoreactivity for IL-1 beta and TNF alpha in human lymphoid and nonlymphoid tissues.

Monoclonal antibodies (MAbs) against two non-cross-reacting antigens of human IL-1 beta (Vhp20 and BRhC3) and human TNF alpha (B154.2 and B154.7) were applied to identify cytokine-containing cells in tissue sections and in cell suspensions. IL-1 beta- or TNF alpha-positive cells were not present in immunostained cytocentrifuge smears prepared from freshly isolated peripheral blood leukocytes, spleen, and lymph node cells. After 18 hours of culture with bacterial endotoxin (LPS), 80% to 90% of blood monocytes, 30% of spleen macrophages, and 2% to 28% of lymph node macrophages were strongly positive for IL-1 beta with either of the MAbs. Furthermore, 25% to 35% of blood monocytes and 6% to 60% of lymph node macrophages were stained for TNF alpha. Cells positive for IL-1 beta or TNF alpha were extremely rare in sections of normal thymus, spleen, and lymph nodes. Immunoreactivity for IL-1 beta or TNF alpha was frequently observed in sections of granulomatous lymphadenitis (N = 11). IL-1 beta or TNF alpha staining was confined to the epithelioid macrophages forming the granuloma, and the intensity of TNF alpha reactivity was generally stronger. The high frequency of cytokine-containing cells in this pathologic condition was confirmed in a cell suspension study showing that 20% of epithelioid macrophages were weakly positive for IL-1 beta and 80% were strongly positive for TNF alpha. The presence of cytokine-containing cells was investigated in cryostat sections of several nonlymphoid organs with normal histologic appearance. IL-1 beta reactivity was not observed in any of the tissues. TNF alpha reactivity was frequently demonstrated in isolated macrophages embedded in the interstitial connective tissue.     

Modification of lipopolysaccharide non-responder cells in low cell density culture

Lipopolysaccharide (LPS) fails to induce polyclonal antibody responses in LPS non-responder C3H/HeJ mice in vivo and also in in vitro cultures of 10(7) cells/ml of C3H/HeJ spleen cells. However, this study shows that LPS elicits polyclonal antibody synthesis in low cell density cultures of C3H/HeJ cells by activation of a translational process without an increase in total RNA and messenger RNA synthesis. Polyclonal antibody synthesis by LPS non-responder cells in low cell density culture was significantly increased by LPS in either serum-free medium or medium containing serum. LPS-induced antibody production by LPS non-responder cells in low cell density was not due to the relative elevation of LPS concentrations.