Control of lipoprotein lipase secretion in mouse macrophages

The regulation of secretion of lipoprotein lipase (LPL) was studied in in vitro-derived mouse bone marrow macrophages (BMM), peritoneal exudate and resident macrophages and in the macrophage-like tumor cell line J774.1. BMM in cultures initiated with low concentrations of bone marrow cells (LC-BMC cultures) secrete more LPL per cell than BMM in cultures initiated with high concentrations of bone marrow cells (HC-BMC cultures). The suppressed state of LPL secretion in HC-BMC cultures could be alleviated by the addition of a colony-stimulating factor source (L-cell-conditioned medium; L-CM) onto the culture medium or exchanging the medium of HC-BMC cultures with medium from LC-BMC cultures for short periods (4 h). Addition of L-CM increased LPL secretion also in LC-BMC cultures. Addition of L-CM to fresh culture medium had little or no effect, suggesting that, in addition to requirement for L-CM, optimal expression depended also on factors released by the growing cells, probably providing optimal growth conditions. L-CM enhanced LPL secretion by thioglycollate-elicited peritoneal macrophages and had no effect on LPL secretion by resident peritoneal macrophages. Secretion of LPL from adherent J774.1 cells showed a biphasic effect. Secretion increased with cell density up to the point when growth inhibition was observed. In dense cultures in which cell proliferation was almost arrested, LPL secretion was remarkably suppressed (80-90%). Change of medium of dense cultures to fresh medium or medium conditioned by sparse cultures (for the last 4 h of culture) led to enhancement of LPL secretion to levels similar to those optimally expressed by sparse cultures. L-CM did not enhance LPL secretion from J774.1 cells. Dense cultures of both BMM and J774.1 cells did not contain a stable inhibitor of LPL secretion and medium from sparse cultures did not contain an inducer of LPL secretion. The data suggest that proliferating macrophages secrete large amounts of LPL, whereas in nonproliferating, quiescent cells, this activity is much reduced. L-CM enhances LPL secretion in quiescent BMM and peritoneal exudate cells to levels expressed by proliferating cells. Since this effect is already expressed after a 4 h incubation period, it is not dependent on cell cycling but could be one of the early responses to this macrophage mitogen. In J774.1 cells, a change of medium is a sufficient signal for enhancement of LPL secretion in quiescent cells.     

Macrophage lineage switching of murine early pre-B lymphoid cells expressing transduced fms genes.

fms genes encoding either wild-type or constitutively activated colony-stimulating factor 1 receptors (CSF-1R) were introduced by retroviral infection into long-term mouse lymphoid cultures. Four early pre-B-cell lines transformed by the feline v-fms oncogene underwent spontaneous and irreversible differentiation to macrophages when transferred from RPMI 1640 to Iscove modified Dulbecco medium. Expression of wild-type human CSF-1R in early pre-B cells conferred no proliferative advantage unless human CSF-1 was added to the culture medium. A clonal, factor-dependent early pre-B-cell line (D1F9), selected for continuous growth on NIH 3T3 cell feeder layers producing human CSF-1, could be maintained in RPMI 1640 medium containing interleukin-7 (IL-7) but also differentiated to macrophages when grown in Iscove modified Dulbecco medium containing human CSF-1. The macrophages retained parental immunoglobulin gene rearrangements and proviral insertions, lost B-cell antigens, expressed butyrate esterase and MAC-1, were actively phagocytic, and no longer survived in IL-7. Unlike factor-independent v-fms transformants, the irreversible commitment of D1F9 cells to differentiate in the macrophage lineage could be suppressed by IL-7, depended on human (but not mouse) CSF-1, and was inhibited by an antibody to human CSF-1R. Signals mediated by transduced CSF-1R can therefore play a deterministic role in cell differentiation.     

Cachectin/tumor necrosis factor and interleukin-1 show different modes of combined effect on lipoprotein lipase activity and intracellular lipolysis in 3T3-L1 cells

Cachectin/tumor necrosis factor (cachectin/TNF) and interleukin-1 (IL-1) share many effects in various tissues and cells, including suppression of lipoprotein lipase (LPL) activity and enhancement of intracellular lipolysis. A possible interaction between cachectin/TNF and IL-1 in these lipase systems was studied in 3T3-L1 adipocytes. The two cytokines showed marked synergy in their suppression of LPL activity in these adipocytes. The least effective dose of cachectin/TNF or IL-1 was at around 5 x 10(-11) or 2.5 x 10(-12) M, respectively. However, when present in combination in amounts as small as 1/20 or 1/100 of the minimum effective dose for either cytokine alone (2.5 x 10(-12) M cachectin/TNF and 2.5 x 10(-14) M IL-1), the cytokines showed marked suppression of LPL activity. In marked contrast, such synergism was not seen for enhancement of intracellular lipolysis. This discrepancy in the combined effects of the two monokines on the two different enzyme systems in the same cells suggests that synergism between cachectin/TNF and IL-1 is unlikely at the level of their receptors on the surface of 3T3-L1 cells. Because the two monokines are considered to be secreted from macrophages under similar conditions, their effect on LPL suppression in many pathophysiological situations would be much greater than that of either monokine alone.     

Opposite effect of linearly polarized light on biosynthesis of interleukin-6 in a human B lymphoid cell line and peripheral human monocytes

The effects of linearly polarized light (LPL) and diffuse light (DL) on the in vitro interleukin-6 (IL-6) production in a human B lymphoma cell line (BMNH) and peripheral monocytes of healthy volunteers were compared. Our data show that there was a significant increase of IL-6 and IgM production in BMNH after exposure to LPL. The increase in IgM secretion was a consequence of its autocrine regulation by IL-6, since in the presence of anti-IL-6 and anti-IL-6 receptor antibodies the LPL-induced IgM secretion was abolished. In contrast to the stimulatory effect on B cells, exposure of human mononuclear phagocytes to LPL markedly reduced the production of IL-6 induced by subsequent stimulation of cells with bacterial endotoxin (LPS). The inhibition as most pronounced when suboptimal doses of LPS were applied. Under identical experimental conditions, DL had no effect on the IL-6 and IgM production of either B cells or monocytes.     

Differential biological activities of human interleukin-1 alpha and interleukin-1 beta

We examined the biological effects induced by both human recombinant interleukin-1 alpha (IL-1 alpha) and beta (IL-1 beta) in five different cell types of human, rat and mouse origin. IL-1 alpha and beta preparations were standardized in terms of biological activity in the EL-4/CTLL bioassay and, in parallel, employed to stimulate PGE2 secretion in human fibroblasts, mesangial cells (MC), C57B1/6 mouse MC, DBA/2 mouse macrophages and Sprague Dawley rat MC. In addition, the co-mitogenic effects of IL-1 alpha and beta were determined in freshly prepared Sprague Dawley rat thymocytes. No significant differences in IL-1 alpha and beta concentration dependent PGE2 production were detectable in the different cell types (MC, fibroblasts and macrophages) of human or mouse origin. Incubation of Sprague Dawley rat MC with both IL-1 alpha and beta resulted in a concentration dependent production of PGE2. However, in contrast to mouse or human MC the potency of IL-1 beta to induce PGE2 in Sprague Dawley rat MC was 26-fold higher compared to IL-1 alpha. In addition, the potency of IL-1 beta to enhance co-stimulated proliferation of Sprague Dawley thymocytes was 200-fold higher than that of equal biological activities of IL-1 alpha. When we tested the additive effects on Sprague Dawley cells, increasing IL-1 beta concentrations added to a fixed IL-1 alpha concentration resulted in a cumulative rise in both, PGE2 secretion by MC and thymocyte proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lymphokine control of type 2 antigen response. IL-10 inhibits IL-5- but not IL-2-induced Ig secretion by T cell-independent antigens.

A supernatant derived from the Th2 clone D10.G4.1 (D10 supernatant) stimulated high numbers of Ig-secreting cells when added to dextran-conjugated anti-delta-antibody (anti-delta-dextran)-activated B cells but stimulated only marginal Ag-specific responses when added to B cells cultured with TNP-Ficoll. When anti-IL-10 antibody was added to cultures containing D10 supernatant, IL-5, and TNP-Ficoll, there was a significant increase in the numbers of anti-TNP-antibody producing cells, suggesting that at least a part of the inhibitory activity of D10 supernatant is mediated by IL-10. Addition of rIL-10 inhibited both TNP-Ficoll- and anti-delta-dextran-mediated Ig secretion that was stimulated in the presence of IL-5 but had no suppressive effect on IL-2-stimulated responses, indicating that its inhibitory effect was selective for a specific mode of B cell activation. Addition of IL-10 did not, however, inhibit anti-delta-dextran-stimulated B cell proliferation. The IL-10-induced-inhibition of Ig secretion was not due to suppression of IFN-gamma production, because the addition of IFN-gamma did not reverse the inhibition, nor did the addition of anti-IFN-gamma mimic the IL-10-mediated inhibition. These data suggest that a composite of lymphokines secreted by Th cells may contain both inhibitory and stimulatory activities. Sorting out the conditions under which stimulation or inhibition is seen may reveal additional diversity in Ag-stimulated pathways of B cell activation.     

Lymphokine-induction of memory B-cell differentiation: differential stimulation of large virgin and memory B-cell differentiation

In order to compare and contrast the requirements of virgin and memory B cells for B-cell differentiation factors, a model system was developed in which low-density rat B cells isolated from 4-week primed antigen-draining lymph nodes were cultured in vitro. This large low-density cell population contained B cells which were 90% surface IgM positive and 60% IgD positive and showed moderately elevated Ia staining. When the cell population was stimulated with antigen plus lymphokines or lymphokines alone, antigen-specific IgG antibody was secreted; this was used as a measure of memory cell differentiation. When the cell population was stimulated with mitogen (lipopolysaccharide plus dextran sulfate) plus lymphokines, polyclonal IgG and IgM secretion was seen and was used as a measure of virgin B-cell differentiation. Using this system, we found that lymphokines contained in a Con A-induced rat spleen cell supernatant (CSN) were sufficient to drive both memory and virgin B-cell differentiation. In contrast, lymphokines contained in the supernatant from the murine T-cell hybridoma B151K12 (B151CFS) were able to induce large amounts of polyclonal IgM and IgG secretion but did not support memory B-cell differentiation. When recombinant human IL-2 was added to these cultures, it acted synergistically to augment virgin B-cell differentiation, but this combination of lymphokines was still not able to support memory B-cell differentiation. Furthermore, recombinant rat interferon-gamma and a commercial source of human BCGF, with or without IL-2, were unable to promote significant virgin or memory B-cell differentiation. These data support the hypothesis that memory B cells and virgin B cells differ in their lymphokine requirements for differentiation into antibody-secreting cells.     

Immunomodulating effects in vitro of interleukin-2 and interferon-gamma on human blood and bone marrow mononuclear cells

Mononuclear cells (MNC) derived from peripheral blood (PBMNC) of 23 normal donors and 4 AIDS patients, and from bone marrow (BMMNC) of 15 normal donors were incubated at 37 degrees C in culture medium alone or in the presence of either natural or recombinant human interleukin-2 (IL-2) or recombinant human interferon-gamma (IFN-gamma; 1-1,000 U/ml). The cultured cells were washed on days 1, 4 or 7 and tested for various immune functions in vitro and for cell surface phenotype. IL-2, but not IFN-gamma, was found mitogenic for both PBMNC and BMMNC. The natural killer (NK) activity of both PBMNC and BMMNC was the only function tested that was markedly augmented (over 100-fold compared to medium control) by both lymphokines. Pretreatment of PBMNC with IL-2 at greater than or equal to 10 U/ml profoundly suppressed (up to 90%) various functions, such as mitogenic responses (phytohemmagglutinin, concanavalin A, pokeweed mitogen), allogeneic mixed leukocyte reaction, antibody production and T cell colony formation in agar. In contrast, some BMMNC functions were elevated at low doses of IL-2 and IFN-gamma, and significant suppression of BMMNC was seen only with high doses of IL-2 (greater than or equal to 100 U/ml) and IFN-gamma (1,000 U/ml). IL-2 was by far more effective than IFN-gamma in both the amplification of NK activity and the suppression of most of the other functions. IL-2, but not IFN-gamma, was found to activate/induce suppressor cells and increased the proportion of Leu-2+ (CD8) cells in PBMNC; the suppressive effect was time- and dose-dependent. The IL-2-induced suppression could be diminished by inclusion of anti-IL-2 antibody during the pretreatment phase. Similar suppressive effects were noted in PBMNC from AIDS patients. These findings suggest that: (a) high-dose IL-2 may elicit immunosuppression which can be mediated by nondiscriminative highly cytotoxic cells (i.e. lymphokine-activated killer cells) and/or by noncytotoxic, nonspecific suppressor cells, and (b) that PBMNC respond differently to the lymphokines than do BMMNC.     

Intracellular regulation of lipoprotein lipase in human monocyte-derived macrophages

The intracellular pathway of lipoprotein lipase (LPL) has been examined in human monocyte-derived macrophages in culture. These cells were previously shown to synthesize and constitutively secrete LPL. The secretion is dependent on new enzyme synthesis. 6-d-old human monocytes have stores of mRNA for linear release of LPL up to 24 h. Enzyme activity in cells and in culture medium was almost completely inhibited by 24 h treatment with tunicamycin, an inhibitor of glycosylation. In monensin-treated cells a pronounced increase in enzyme activity was found, whereas the secreted activity was markedly reduced. This indicates that LPL in human monocytes is processed through a pH sensitive part of the Golgi complex and that the terminal glycosylation is not needed for the expression of its catalytic activity. Our results suggest that lysosomal function is not important in secretion of the enzyme, whereas vesicular transport seem to be involved in regulating LPL in human monocyte-derived macrophages in culture.     

Effects of human alveolar macrophages on the induction of lymphokine (IL 2)-activated killer cells

Normal human alveolar macrophages (AM) significantly and reproducibly suppress induction of IL 2-activated killer (LAK) cell activity against allogeneic Burkitt's lymphoma (Daudi) cells. Incubation of purified peripheral blood lymphocytes for 4 days with autologous AM and 1 U/ml of IL 2 resulted in AM-mediated suppression of LAK activity, whereas peripheral blood monocytes isolated freshly by centrifugal elutriation from the same donor potentiated induction of LAK activity by IL 2. The suppression of LAK cell induction by human AM was dependent on the density of AM added to the lymphocyte cultures. Recombinant IFN-gamma did not affect AM-mediated suppression of LAK cell induction by IL 2. Both AM and monocytes stimulated with lipopolysaccharide markedly suppressed LAK cell induction by IL 2. AM-mediated down-regulation was seen only when AM were added immediately after the start of incubation of lymphocytes with IL 2; AM potentiated LAK activity when added 1 day later. Similar AM-mediated suppression of LAK cell induction was observed with four lines of allogeneic lung cancer cells as targets for LAK activity. These results indicate that AM may be important in regulation of in situ induction of LAK activity in the lung.     

Bacterial lipopolysaccharide inhibits dengue virus infection of primary human monocytes/macrophages by blockade of virus entry via a CD14-dependent mechanism

Monocytes/macrophages (MO/Mphi) are the major target cells for both dengue virus (DV) and bacterial lipopolysaccharide (LPS), and the aim of this study was to define their interactions. We had found that LPS markedly suppressed DV infection of primary human MO/Mphi when it was added to cultures prior to or together with, but not after, viral adsorption. The inhibitory effect of LPS was direct and specific and was not mediated by LPS-induced secretion of cytokines and chemokines such as tumor necrosis factor alpha, interleukin-1beta (IL-1beta), IL-6, IL-8, IL-12, alpha interferon, MIP-1alpha, and RANTES. In fact, productive DV infection was not blocked but was just postponed by LPS, with a time lag equal to one viral replication cycle. Time course studies demonstrated that LPS was only effective in suppressing DV infection of MO/Mphi that had not been previously exposed to the virus. At various time points after viral adsorption, the level of unbound viruses that remained free in the culture supernatants of LPS-pretreated cultures was much higher than that of untreated controls. These observations suggest that the LPS-induced suppression of DV replication was at the level of virus attachment and/or entry. Blockade of the major LPS receptor, CD14, with monoclonal antibodies MY4 or MoS39 failed to inhibit DV infection but could totally abrogate the inhibitory effect of LPS. Moreover, human serum could significantly enhance the LPS-induced DV suppression in a CD14-dependent manner, indicating that the "binding" of LPS to CD14 was critical for the induction of virus inhibition. Taken together, our results suggest that LPS blocked DV entry into human MO/Mphi via its receptor CD14 and that a CD14-associated cell surface structure may be essential for the initiation of a DV infection.     

Macrophage population dynamics within fetal mouse fibroblast cultures derived from C57BL/6, CD-1, CF-1 mice and interleukin-6 and granulocyte colony stimulating factor knockout mice

In vitro models of macrophage growth, differentiation, and function are needed to facilitate the study of their biology as important immune facilitator cells and as frequent targets of bacterial and viral infection. A simple method for the selective expansion and continuous culture of mouse macrophages from primary explant cultures of mouse embryonic tissue is described. Culture in Dulbecco modified Eagle medium (DMEM) low-glucose (1 g/L) formulation (DMEM/L) inhibited fibroblast growth. In contrast, macrophages continued to proliferate in the presence of DMEM/L when in contact with the fibroblasts. Alternating growth in high-glucose DMEM with DMEM/L produced a 1.16- to 2.1-fold increase (depending on mouse strain) in the percentage of macrophages within the cell culture in comparison with culturing in DMEM with high glucose exclusively. Macrophage yields of over 1 million cells/T12.5 flask were achieved by passages 3-4, and, thereafter, declined over the next 5-10 passages. The peak percentage of macrophages within a culture varied depending on the strain of mouse (C57BL/6, CD-1, and CF-1 and two knockout C57BL/6 strains deficient in either interleukin-6 [IL-6] or granulocyte colony stimulating factor [GCSF]). The GCSF (-/-)-derived cultures had the lowest peak macrophage content (30%) and CD-1 the highest content (64.9%). The IL-6 (-/-) and CD-1 cultures appeared to spontaneously transform to create cell lines (IL6MAC and CD1MAC, respectively) that were composed of 50-75% macrophages. The macrophages were phagocytic and were positive for CD14, acetylated low-density lipoprotein receptors, and F4-80 antigen. Light and electron microscopy showed that the cultured macrophages had in vivo-like morphological features, and they could be plated to high purity by differential attachment to petri dishes in serum-free medium.     

Frequency analysis of lymphokine-secreting CD4+ and CD8+ T cells activated in a graft-versus-host reaction

Lymphokine secretion by in vivo-activated T cells was analyzed at the population and single-cell levels in lymphocytes from mice undergoing an acute allogeneic graft-vs-host reaction (GVHR). Three observations were made. First, constitutive lymphokine production by these cells was very low but could be dramatically up-regulated by TCR ligation. Thus, even when harvested at the peak of the GVHR, fewer than 0.1% of lymphocytes secreted detectable granulocyte-macrophage (GM)-CSF, IFN-gamma, or IL-3 in the first 24 h in vitro, and average production of these lymphokines in bulk cultures was less than 10(-5) U/cell. However, when cultured for 24 h with anti-CD3 antibody under conditions which activated less than 0.1% of normal cells, about 30% of GVHR T cells secreted GM-CSF, IFN-gamma, and/or IL-3, and average production levels were increased by 10(3)- to 10(4)-fold. Together with evidence that host alloantigen-induced lymphokine secretion was 10 to 100 times lower than the anti-CD3 response, these data suggest that physiologic lymphokine synthesis by most T cells is low (less than 10(-18) mol of IL-3 per cell) but can be raised above the threshold of detection by TCR cross-linking. Second, individual GVHR lymphocytes varied markedly in their total and relative production of different lymphokines in response to anti-CD3 stimulation, with some cells secreting IL-3 alone, some secreting IL-3 accompanied by other lymphokines (GM-CSF and/or IFN-gamma), and some secreting other lymphokines without detectable IL-3. Finally, both CD4+ and CD8+ T cells from GVHR mice responded to anti-CD3 antibody by secreting IL-3 and other lymphokines: purified CD4+ cells contained an average of 16% and CD8+ cells an average of 10% anti-CD3-inducible lymphokine-secreting cells. By contrast, only 2 to 3% of cells of either subset formed clones in cultures with host allogeneic cells and IL-2, suggesting that clonogenic alloreactive cells were a minority of the T cells activated in the GVHR.     

Suppression of endothelial or lipoprotein lipase in THP-1 macrophages attenuates proinflammatory cytokine secretion

LPL and endothelial lipase (EL) are associated with macrophages in human atherosclerotic lesions, and overexpression of LPL in mouse macrophages is associated with a greater extent of atherosclerosis. To investigate potential mechanisms by which macrophage-derived lipase expression may mediate proatherogenic effects, we used lentivirus-mediated RNA interference to suppress the expression of either LPL or EL within THP-1 macrophages. After suppression of either LPL or EL, significant decreases in the concentration of interleukin-1beta, interleukin-6, monocyte chemoattractant protein-1, and tumor necrosis factor-alpha were observed. Incubation of THP-1 macrophages with either mildly or extensively oxidized LDL consistently decreased cytokine expression, which was additive to that contributed by lipase suppression. Decreased lipase expression was also associated with an altered lipid composition, with reduced percentages of cholesterol (unesterified and esterified), triglycerides, and lysophosphatidylcholine. Microarray data indicated a decreased expression of proinflammatory genes, growth factors, and antiapoptotic genes. By contrast, there was an increased expression of lipoprotein receptors (scavenger receptor 1, low density lipoprotein receptor, scavenger receptor class B type I, and CD36). Thus, we conclude that the suppression of either LPL or EL decreases proinflammatory cytokine expression and influences the lipid composition of THP-1 macrophages. These results provide further insight into the specific metabolic and potential pathological roles of LPL and EL in human macrophages.     

The effects of soluble products of activated lymphocytes and macrophages on blastocyst implantation events in vitro

The purpose of this study was to investigate the effects of soluble products of activated lymphocytes and macrophages on mouse blastocyst attachment and trophoblast outgrowth in vitro. Hatched blastocysts were incubated with medium alone, supernatant fluids from mixed lymphocyte cultures (MLC), and with individual human and murine lymphokines and monokines in fibronectin-coated wells. Cultures were assessed at 24, 48, and 72 h for blastocyst attachment and trophoblast outgrowth. Both human and murine MLC supernatant fluids significantly enhanced trophoblast outgrowth in vitro. The cytokine, interleukin-1 beta (Il-1 beta), at a concentration of 10(3) U/ml, inhibited blastocyst attachment but significantly enhanced trophoblast outgrowth of attached blastocysts. Granulocyte, macrophage-colony-stimulating factor (GM-CSF) at a concentration of 250 U/ml significantly inhibited blastocyst attachment, while gamma interferon (gamma-IFN) at a concentration of 2.5 x 10(3) U/ml significantly inhibited trophoblast outgrowth and caused degenerative morphological changes in these cells. The results of this study indicate that products of activated immune cells may either facilitate or impede implantation events depending on the types of predominant cytokines present, their concentration(s), and the timing of their secretion relative to embryonic development.     

Splenic macrophages enhance prolactin-induced progestin secretion from mature rat granulosa cells in vitro.

The effect of splenic macrophages on in vitro progestin secretion (the sum of the progesterone and 4-pregnen-20 alpha-ol-3-one concentrations in the medium) from mature rat granulosa cells was examined by means of co-culture techniques. When splenic macrophages (3.0 x 10(5) cells/ml) obtained from adult female rats on the evening of proestrus (1800 h) were added to granulosa cells (1.5 x 10(5) cells/ml) and co-cultured for 96 h in the absence of prolactin (PRL), progestin secretion from granulosa cells did not change. However, co-culture of granulosa cells with the macrophages in the presence of PRL (2 micrograms/ml) significantly enhanced progestin secretion after 48 h of culture. This stimulatory effect on progestin secretion was observed only when the number of macrophages added was more than twice the number of granulosa cells. On the other hand, splenic macrophages obtained on the evening of diestrus had no effect on progestin secretion from granulosa cells even in the presence of PRL. These results suggest that splenic macrophages can enhance PRL action so as to stimulate progestin secretion from granulosa cells and that this function of splenic macrophages varies during the estrous cycle.     

Effect of berberrubine on interleukin-8 and monocyte chemotactic protein-1 expression in human retinal pigment epithelial cell line

We examined the effects of berberrubine, a protoberberine alkaloid, on interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) expression in a human retinal pigment epithelial cell line (ARPE-19) stimulated with interleukin-1beta (IL-1beta) or tumor necrosis factor alpha (TNF-alpha). ARPE-19 cells were cultured to confluence. Berberrubine and IL-1beta or TNF-alpha were added to the medium. IL-8 and MCP-1 protein concentrations were measured using enzyme-linked immunosorbent assay. IL-8 and MCP-1 mRNA were measured by real time polymerase chain reaction. Nuclear factor kappaB (NF-kappaB) translocation was examined by immunofluorescent staining/microscopy. Berberrubine dose-dependently inhibited IL-8 and MCP-1 protein levels in the media and mRNA expression of the cells stimulated with IL-1beta or TNF-alpha. Immunofluorescent staining/microscopy of NF-kappaB in the nucleus of unstimulated cells was faint (51+/-14 arbitrary units). Fluorescein was dense (215+/-42 or 170+/-24 arbitrary units, respectively) 30 min after stimulation with IL-1beta or TNF-alpha and was decreased to 62+/-18 or 47+/-16 arbitrary units, respectively, by berberrubine. Berberrubine dose-dependently inhibited IL-8 and MCP-1 expression and protein secretion induced by IL-1beta or TNF-alpha. Possibly, the effect on chemotactic factors may be via suppression of NF-kappaB translocation.     

Immunosuppression induced by attenuated Salmonella. Reversal by IL-4

We previously demonstrated that an aroA- strain of Salmonella typhimurium, which provides excellent protection against virulent Salmonella challenge, also rendered immunized mice unable to mount in vivo and in vitro antibody responses to heterologous Ag. Coculture studies using transwell plates indicated that suppression was mediated by soluble factors. The suppressive cells were identified as belonging to the monocytic linkage. Macrophage precursors as well as mature adherent macrophages mediated the observed suppression. In the present study, the mechanism of immunosuppression was investigated. Suppression was found to be genetically nonrestricted as spleen cells from immunized C3HeB/FeJ mice (H-2k) suppressed the anti-SRBC plaque-forming cell (PFC) responses of normal spleen cells from two MHC noncompatible mouse strains, BALB/c (H-2d) and C57BL/6 (H-2b). Time course experiments demonstrated that the addition of spleen cells from immunized mice to normal splenocytes as late as day 4 of a 5-day assay was still markedly suppressive. Furthermore, suppression of the PFC responses was accompanied by a profound inhibition of the capacity of immune splenocytes to produce IL-2 in response to in vitro stimulation by Con A. Coculture studies showed that immune spleen cells were able to suppress IL-2 production by normal splenocytes in a dose-dependent fashion. However, the suppressed PFC responses of immune spleen cells could not be reversed by the exogenous addition of up to 200 U/ml of IL-2, suggesting that immune splenocytes are also defective in their ability to respond to IL-2. In marked contrast, suppression of PFC responses was reduced by more than 50% by the addition of as little as 1 U/ml of IL-4 and was completely abrogated when 5 U/ml of IL-4 were added to in vitro cultures of spleen cells from immunized mice. The antisuppressive action of IL-4 appeared to be via its inhibitory effect on activated macrophages. The implications of the above findings are discussed.     

Cytokine Regulation of Human Immunodeficiency Virus Type 1 Entry and Replication in Human Monocytes/Macrophages through Modulation of CCR5 Expression

Human macrophages express chemokine receptors that act as coreceptors for human immunodeficiency virus type 1 (HIV-1) and are major targets for HIV-1 infection in vivo. The effects of cytokines on HIV-1 infection of macrophages and on the expression of CCR5, the principal coreceptor for macrophage-tropic viruses, have now been investigated. Expression of CCR5 on the surface of freshly isolated human monocytes was virtually undetectable by flow cytometry with the monoclonal antibody 5C7. However, after culture of monocytes for 48 h in serum-free medium, approximately 30% of the resulting macrophages expressed CCR5 and the cells were susceptible to infection by macrophage-tropic HIV-1. Addition of either macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) to the cultures markedly increased both the extent of HIV-1 entry and replication as well as surface expression of CCR5. In contrast, addition of the T-helper 2 (Th2) cell-derived cytokine interleukin-4 (IL-4) or IL-13 prevented the expression of CCR5 induced by culture in medium alone, and IL-4 inhibited virus entry, replication, and cytopathicity under these conditions. IL-4 or IL-13 also prevented the stimulatory effects of M-CSF or GM-CSF on CCR5 expression as well as HIV-1 entry and replication. In addition, IL-4 reversed the increase in CCR5 expression induced by pretreatment of cells with M-CSF. Although IL-10 also inhibits HIV-1 replication in macrophages, it did not suppress surface CCR5 expression induced by colony-stimulating factors. These results indicate that the cytokine environment determines the susceptibility of macrophages to HIV-1 infection by various mechanisms, one of which is the regulation of HIV-1 coreceptor expression.