Plasminogen activator production by human monocytes. I. Enhancement by activated lymphocytes and lymphocyte products.

Human monocytes, but not nylon wool column-nonadherent lymphocytes, produce plasminogen activator. The activity is found only in association with intact cells. Exposure of monocytes to activated lymphocytes or to lymphokine-rich supernatants enhances monocyte plasminogen activator production. The assay allows assessment of baseline and activated human monocyte function.     

Phospholipid synthesis by activated human B lymphocytes

Pokeweed mitogen- (PWM) stimulated DNA and Ig synthesis in human B cells is dependent on the presence of T cells and adherent cells, but the influence of these regulatory cells on earlier activation events is unknown. We have studied the T cell and monocyte influence on the incorporation of [methyl-14C]choline chloride into B cell phospholipids (PL) after varying periods of in vitro culture with or without pokeweed mitogen (PWM). By separating B and T cells after choline pulsing, a peak in PWM-induced PL synthesis of B cells at days 1 to 2 was revealed, whereas the T cell response was later (days 2 to 3). In the first 4 hr of culture, the purified B cell plus monocyte fraction incorporated choline four to six times faster than the T cell fraction, but PWM did not increase choline incorporation, whether these fractions were cultured separately or together. When cultures were pulsed with choline between 16 and 20 hr with or without PWM, monocytes incorporated choline six to nine times faster than T cells, and B cells were intermediate. Also at 16 to 20 hr of culture, a significant PWM-induced increase in choline incorporation by B cells was evident and was dependent on the presence of T cells and monocytes. The monocytes showed no increased choline incorporation due to PWM. Thus, the influence of regulatory cells on the PWM response in B cells is evident within the first 24 hr.     

Modulation of interleukin 1 production by activated macrophages: in vitro action of hydrocortisone, colchicine, and cytochalasin B

In vitro cultured irradiated rat peritoneal macrophages (XPCs) secreted markedly more Interleukin 1 (IL-1) when pretreated with carrageenan. Exposure of cultures of carrageenanpretreated as well as untreated XPCs to hydrocortisone (OHC) produced a dose-dependent inhibition of IL-1 secretion. OHC at concentrations from 10^?4 to 10^?6M completely blocked the enhanced IL-1 secretion induced by carrageenan pretreatment. These observations suggest that the complex immunosuppressive effects of glucocorticoids could partly result from their capability to control IL-1 production. Colchicine, a microtubule depolymerizing agent, markedly enhanced IL-1 secretion in cultures of both carrageenan-pretreated and untreated XPCs. Stimulatory effects were dose dependent and maximum stimulation occurred at 10^?4M, i.e., at a concentration known to profoundly affect the microtubular function. Lumicolchicine, however, was inactive. Cytochalasin B, a microfilament disrupting agent and inhibitor of phagocytosis, elicited a dose-dependent increase of IL-1 secretion in cultures of carrageenanactivated XPCs but was inhibitory for unstimulated XPCs. Although mechanisms by which Colchicine and Cytochalasin B act in stimulating IL-1 production remain unclear, our study demonstrates that production and secretion of IL-1 do not require participation of intact components of the cytoskeleton.     

Activation of B lymphocytes with human serum-treated zymosan

C3 fragments fixed on zymosan particles were presented to resting human B lymphocytes. The opsonized zymosan (Ops-Z) particles induced release of leukocyte migration inhibitory factor, a slight decrease in mIgD, and a slight increase in the activation marker Blast-2. The B cells did not proceed further along the pathway of activation: they did not respond to B cell growth factor (BCGF) and Ops-Z did not synergize with other activators for BCGF response either. Thus, we found that interaction between C3 fragments and CR2 initiates the activation of human B lymphocytes, but this is limited to the early phase.     

Activation of Fc receptor-bearing lymphocytes by immune complexes. I. Stimulation of lymphokine production by nonadherent human peripheral blood lymphocytes

Activation of human peripheral blood lymphocytes by incubation with particulate immune complexes or aggregated human gamma-globulin was studied by measuring the release of leukocyte migration inhibitory factor (LIF) activity. LIF-active supernatants were consistently produced when nonadherent lymphocytes containing less than 1% surface immunoglobulin-bearing cells and less than 0.2% nonspecific esterase-positive monocytes were incubated in the presence of RBC sensitized with rabbit or human antibodies or with pooled heat-aggregated human gamma-globulin. This immune complex-induced lymphokine production (ICLP) was dependent on the presence of cells bearing receptors for the Fc portion of IgG (Fc gamma). ICLP could not be demonstrated with lymphocyte preparations enriched for B cells even though the latter showed vigorous LIF production in the presence of complement-sensitized erythrocytes. ICLP was dependent on the concentration of lymphocytes and of stimulant as well as on the duration of coincubation, and it required active metabolic processes and RNA and protein synthesis but not DNA synthesis. Ca++ but not Mg++ was obligatory. ICLP by non-B Fc gamma receptor-bearing lymphocytes may play a role in antibody-dependent protective inflammation and immunologic injury phenomena, which is similar to that of lymphokine release by antigen-activated T cells in delayed hypersensitivity responses.     

Activated human B lymphocytes express cyclooxygenase-2 and cyclooxygenase inhibitors attenuate antibody production

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of inflammatory diseases and target cyclooxygenases 1 and 2 (Cox-1, Cox-2) that are responsible for PG production. Newer Cox-2-selective drugs have been heavily prescribed to quench inflammation. Little is known about whether or not these drugs influence human B lymphocytes and their ability to produce Ab. We report herein that activated human B cells not only highly express Cox-2 and produce PGs, but that the NSAID indomethacin and Cox-2-selective drugs profoundly inhibit the ability of human B cells to produce IgG and IgM in vitro. Human blood B cells highly express Cox-2 mRNA and protein and produce PGs after activation with CD40L, pansorbin, or CD40L plus BCR engagement. Cox-2 is also highly expressed by human tonsil B cells, as shown by immunohistochemistry. Cox-inhibiting drugs modestly affect purified B cell proliferation but profoundly reduce Ab production. The ability of whole blood to produce IgM and IgG following stimulation is also strongly inhibited. In support that Cox-2 plays a seminal role in B lymphocyte Ab production, Cox-2 knockout mice have 64% less IgM and 35% less IgG than normal littermate controls. These findings support that NSAIDs and the new Cox-2-selective drugs have an unsuspected target, the B cell, and attenuate Ab production in humans. Use of NSAIDs may therefore influence autoantibody production in autoimmune diseases and may dampen humoral immunity in response to antigenic challenge/vaccination.     

Native low-density lipoproteins stimulate leukotriene B4 production by human monocyte-derived macrophages.

We have evaluated the effect of native low-density lipoproteins (LDL) on the production of leukotriene B4 (LTB4), a potent inflammatory and chemotactic factor, by human monocyte-derived macrophages. The capacity of LDL (d, 1.024-1.050 g/ml) to increase LTB4 secretion was dose-dependent with an optimal response at 100 micrograms LDL protein/ml, representing an approx. 7.5-fold stimulation over basal levels at 10 days of culture; the half-maximal response occurred at 20 micrograms/ml. The effect of LDL on LTB4 production was rapid (within 15 min) and was maintained for at least 21 h. The generation of LTB4 in response to LDL was partially inhibited (approx. 70% inhibition) by EDTA (5 mM) and by a monoclonal antibody (IgG-C7; 160 micrograms/ml) directed against the binding site of the cellular LDL receptor. In addition, the effects of native LDL and acetylated LDL were additive. These findings suggest that the specific interaction of LDL with its high affinity receptor represents a major component in the stimulation of the production of LTB4 by human monocyte-derived macrophages.     

Interactions between B lymphocyte subpopulations. Augmentation of the responses of resting B lymphocytes by activated B lymphocytes

Mouse B lymphocytes were fractionated from normal T lymphocyte-depleted spleen cell populations using discontinuous percoll gradients and were stimulated with rabbit F(ab')2 anti-mouse mu-specific antibodies (anti-mu) plus the supernatant of Con A-stimulated rat spleen cells (SN) as a source of lymphokines. The responses of small (mean volume 120 mu 3), dense (greater than 1.087 specific gravity), resting (least spontaneous thymidine incorporation) B lymphocytes were augmented by irradiated (4000 rad), larger (mean volume greater than 170 mu 3), less dense (less than 1.081 specific gravity), activated (greater spontaneous thymidine incorporation) B lymphocytes. Proliferation was augmented 2- to 4-fold and polyclonal antibody-forming cell responses three- to sixfold. Maximal augmentation of the responses of 5 X 10(4) resting B cells was obtained with 10(4) activated B cells. Augmenting activity was specific for activated B lymphocytes in that responses were not augmented by irradiated thymocytes, T lymphoblasts, macrophages, or additional supernatant. B lymphocytes activated in vitro by LPS or anti-mu also had augmenting activity. Augmentation of responses was maximal only when activated B lymphocytes were added simultaneously with anti-mu. The interaction between activated and resting B lymphocytes did not appear to be genetically restricted. Interestingly, the augmenting activity of activated B cells could be reconstituted by a combination of supernatant and cell membranes from these cells but not by either alone, suggesting that two components are required, one soluble and the other membrane-bound. Thus, a functional interaction has been demonstrated between B lymphocyte subpopulations which differ in their state of activation, and this interaction appears to involve a novel mechanism of action.     

Anti-tumor antibody produced by human tumor-infiltrating and peripheral blood B lymphocytes

Cell suspensions from 69 human tumor biopsies and malignant effusions depleted of infiltrating T cells were incubated for 10–14 days with mitomycin-C-treated cells of the transformed T cell line MOT as feeder cells. B lymphocytes proliferated and differentiated as indicated by immunoglobulin (Ig) seerction in the culture supernatants (B cell expansion). Ig was present in culture supernatants of tumor cell suspensions incubated without MOT feeder cells (non-expanded cells), but the addition of MOT feeder cells to these cultures invariably resulted in a significant increase in Ig concentration. While IgG, IgA. and IgM isotypes were all detected in supernatants of both expanded- and nonexpanded tumor cell suspensions, the increase in total Ig induced by MOT feeder cells was mainly due to an increase in IgG. Peripheral blood B lymphocytes (PBBL) from 15 cancer patients and 4 healthy individuals were also successfully expanded by the same method. In these it was shown that IgA was the predominant Ig isotype. Using a modified enzyme-linked immunosorbent assay, IgG of 25/36 expansions from tumor cell suspensions showed reactivity with autologous tumor targets, and that from 10/13 expansions reacted with allogeneic tumor targets of the same histological diagnosis. No reactivity was found against tumor targets of different histology. IgG of 4/10 expansions of PBBL from cancer patients showed reactivity with allogeneic tumor targets of the same histology, while no reactivity was demonstrated against tumor targets of different histology. IgG of expanded PBBL from healthy individuals showed no reactivity against tumor targets. This method allows detailed study of the specific humoral antitumor immune response of intratumoral and peripheral blood B lymphocytes in cancer.Work supported by grants from the Share and Concern Foundations and grant CA MOPP from the National Institutes of Health, C.J.A.P. is a visiting scientist from the University of Nijmegen, Department of Medical Oncology, Nijmegen, The Netherlands, and is supported by a Fulbright Senior Research Grant and grants from the Dutch Cancer Society and the Regional Cancer Center of the East Netherlands (IKO). J.A.M.B. is a visiting scientist from the University of Sao Paolo, Department of Immunology, Brazil, and is supported by grant 90/1844-4 from the FAPESP     

IL-10 inhibits cytokine production by activated macrophages

IL-10 inhibits the ability of macrophage but not B cell APC to stimulate cytokine synthesis by Th1 T cell clones. In this study we have examined the direct effects of IL-10 on both macrophage cell lines and normal peritoneal macrophages. LPS (or LPS and IFN-gamma)-induced production of IL-1, IL-6, and TNF-alpha proteins was significantly inhibited by IL-10 in two macrophage cell lines. Furthermore, IL-10 appears to be a more potent inhibitor of monokine synthesis than IL-4 when added at similar concentrations. LPS or LPS- and IFN-gamma-induced expression of IL-1 alpha, IL-6, or TNF-alpha mRNA was also inhibited by IL-10 as shown by semiquantitative polymerase chain reaction or Northern blot analysis. Inhibition of LPS-induced IL-6 secretion by IL-10 was less marked in FACS-purified peritoneal macrophages than in the macrophage cell lines. However, IL-6 production by peritoneal macrophages was enhanced by addition of anti-IL-10 antibodies, implying the presence in these cultures of endogenous IL-10, which results in an intrinsic reduction of monokine synthesis after LPS activation. Consistent with this proposal, LPS-stimulated peritoneal macrophages were shown to directly produce IL-10 detectable by ELISA. Furthermore, IFN-gamma was found to enhance IL-6 production by LPS-stimulated peritoneal macrophages, and this could be explained by its suppression of IL-10 production by this same population of cells. In addition to its effects on monokine synthesis, IL-10 also induces a significant change in morphology in IFN-gamma-stimulated peritoneal macrophages. The potent action of IL-10 on the macrophage, particularly at the level of monokine production, supports an important role for this cytokine not only in the regulation of T cell responses but also in acute inflammatory responses.     

Activation of human B lymphocytes. III. Concanavalin A-induced generation of suppressor cells of the plaque-forming cell response of normal human B lymphocytes.

Con A-induced suppression of the direct PFC response to polyclonal stimulation in human B cells has been described. Two types of experiments are presented. First, Con A was added directly to PWM-stimulated PB or tonsil cells resulting in a dose-dependent suppression of the PFC response, with maximal suppression occurring at a Con A concentration of 10 mug/ml. This suppression is completely removed by the simultaneous addition of alphaMM to the cultures. Secondly, Con A stimulation of tonsil or PB lymphocytes generated a population of cells which when added to autologous lymphocyte cultures induced a marked and reproducible suppression of the PFC response. The generation of suppressor cells is dependent on cell division and is blocked by alpha MM. Once generated the process of suppression is indpendent of the presence of Con A itself and is mediated by an activated lymphocyte population. These studies demonstrate a simple and reproducible model for the generation of a population of suppressor cells capable of inhibiting the direct PFC response to PWM-induced polyclonal activation of normal human B lymphocytes.