Monocyte chemoattractant protein-1 selectively inhibits the acquisition of CD40 ligand-dependent IL-12-producing capacity of monocyte-derived dendritic cells and modulates Th1 immune response

Accumulating evidence indicates that monocyte chemoattractant protein-1 (MCP-1), a CC chemokine, also displays immunoregulatory functions and may be involved in Th subset differentiation. In this study, we examined the effects of MCP-1 on the cytokine-driven differentiation of monocytes into dendritic cells (DCs), the most potent APCs for naive T cells. We found that DCs generated in the presence of MCP-1 displayed a markedly reduced production of IL-12 in response to CD40 ligand but not in response to Staphylococcus aureus stimulation in the presence or absence of IFN-gamma. The production of IL-10, a potent endogenous IL-12 inhibitor, was not affected by MCP-1. Whereas the inhibitory activity of MCP-1 on IL-12 production by monocytes was sensitive to pertussis toxin, its effects on DC differentiation were pertussis toxin resistant. MCP-1 did not affect the surface phenotype and T cell-stimulating activity of DCs, but most interestingly, naive T cells stimulated with MCP-1-primed DCs produced much less IFN-gamma but the same levels of IL-13. Taken together, our results indicated that MCP-1 modulates the differentiation of monocytes into DCs and may thereby inhibit Th1 cell development.     

Interferon-gamma Stimulates Monocyte Chemotactic Protein-1 Expression by Monocytes

Monocyte chemotactic protein (MCP-1) is a specific monocyte chemoattractant and activating factor produced by both immune cells (mononuclear phagocytes and lymphocytes) and non-immune cells (parenchymal and stromal cells). In order to define the conditions under which human monocytes express MCP-1, monocytes were exposed to IFN-gamma, IL- lbeta, TNF-alpha, IL-4 or PHA under serum free conditions. There was no significant MCP-1 production by monocytes following exposure to IL-lbeta, TNF-alpha or IL-4. In contrast, stimulation with IFN-gamma resulted in a dose dependent increase in MCP-1 protein and mRNA expression. Simultaneous stimulation with IFN-gamma and IL-1beta or TNF-alpha resulted in no further increase in MCP-1 production. It is concluded that IFN-gamma, primarily a product of T(H)1 T lymphocytes, stimulates the expression of MCP-1 by monocytes.     

Expression and function of C5a receptor in mouse microvascular endothelial cells

The complement-derived anaphylatoxin, C5a, is a potent phlogistic molecule that mediates its effects by binding to C5a receptor (C5aR; CD88). We now demonstrate specific binding of radiolabeled recombinant mouse C5a to mouse dermal microvascular endothelial cells (MDMEC) with a K(d50) of 3.6 nM and to approximately 15,000-20,000 receptors/cell. Recombinant mC5a competed effectively with binding of [(125)I]rmC5a to MDMEC. Enhanced binding of C5a occurred, as well as increased mRNA for C5aR, after in vitro exposure of MDMEC to LPS, IFN-gamma, or IL-6 in a time- and dose-dependent manner. By confocal microscopy, C5aR could be detected on surfaces of MDMEC using anti-C5aR Ab. In vitro expression of macrophage inflammatory protein-2 (MIP-2) and monocyte chemoattractant protein-1 (MCP-1) by MDMEC was also measured. Exposure of MDMEC to C5a or IL-6 did not result in changes in MIP-2 or MCP-1 production, but initial exposure of MDMEC to IL-6, followed by exposure to C5a, resulted in significantly enhanced production of MIP-2 and MCP-1 (but not TNF-alpha and MIP-1alpha). Although LPS or IFN-gamma alone induced some release of MCP-1 and MIP-2, pre-exposure of these monolayers to LPS or IFN-gamma, followed by addition of C5a, resulted in synergistic production of MIP-2 and MCP-1. Following i.v. infusion of LPS into mice, up-regulation of C5aR occurred in the capillary endothelium of mouse lung, as determined by immunostaining. These results support the hypothesis that C5aR expression on MDMEC and on the microvascular endothelium of lung can be up-regulated, suggesting that C5a in the co-presence of additional agonists may mediate pro-inflammatory effects of endothelial cells.     

G(i)-protein-dependent inhibition of IL-12 production is mediated by activation of the phosphatidylinositol 3-kinase-protein 3 kinase B/Akt pathway and JNK

Ligands for certain G(i)-protein-coupled receptors (GiPCRs) potently inhibit the production of IL-12 by human monocytes. We addressed the intracellular signaling mechanisms by which this occurs using primary human cells. Stimulation with the GiPCR ligands C5a and 1-deoxy-1-[6-[(3-iodophenyl)methyl]amino]-9H-purine-9-y1]-N-methyl-beta-D-ribofuranuronamide (IB-MECA) blocked the production of IL-12 p70 by human monocytes stimulated with LPS and IFN-gamma. In addition, C5a reduced the expression of mRNA for IL-12 p35, p40, IL-23 p19, and IL-27 p28. This effect was due neither to a down-regulation of TLR4 or IFN-gamma receptor on the cell surface nor to interference with IFN-gamma signaling, because IFN-gamma-induced up-regulation of HLA-DR and CD40 were unaffected. C5a or IB-MECA activated the PI3K/Akt signaling pathway and induced the phosphorylation of the MAPK p38, ERK, and JNK. Inhibition of the PI3K/Akt signaling pathway with wortmannin or an inhibitor of Akt activity, and inhibition of JNK but not ERK prevented IL-12 and IL-23 suppression by C5a. These data extend observations on IL-12 suppression by C5a to IL-23 and IL-27, and are the first to demonstrate the intracellular signaling events leading to IL-12 and IL-23 inhibition after GiPCR activation.     

CD40 ligand-activated human monocytes amplify glomerular inflammatory responses through soluble and cell-to-cell contact-dependent mechanisms.

Monocytes/macrophages play a critical role in the initiation and progression of a variety of glomerulonephritides. We sought to define the interactions between physiologically activated human monocytes and glomerular mesangial cells (MC) by employing a cell culture system that permits the accurate assessment of the contribution of soluble factors and cell-to-cell contact. Human peripheral blood monocytes, primed with IFN-gamma and GM-CSF, were activated with CD40 ligand (CD40L) or TNF-alpha and cocultured with MC. CD40L-activated monocytes induced higher levels of IL-6, monocyte chemoattractant protein-1 (MCP-1) and ICAM-1 synthesis by MC. Separation of CD40L-activated monocytes from MC by a porous membrane decreased the mesangial synthesis of IL-6 by 80% and ICAM-1 by 45%, but had no effect on MCP-1. Neutralizing Abs against the beta 2 integrins, LFA-1 and Mac-1, decreased IL-6 production by 40 and 50%, respectively. Ligation of mesangial surface ICAM-1 directly enhanced IL-6, but not MCP-1, production. Simultaneous neutralization of soluble TNF-alpha and IL-1 beta decreased MCP-1 production by 55% in membrane-separated cocultures of MC/CD40L-activated monocytes. Paraformaldehyde-fixed CD40L-activated monocytes (to preserve membrane integrity but prevent secretory activity), cocultured with MC at various ratios, induced IL-6, MCP-1, and ICAM-1 synthesis by MC. Plasma membrane preparations from activated monocytes also induced mesangial IL-6 and MCP-1 synthesis. The addition of plasma membrane enhanced TNF-alpha-induced mesangial IL-6 production by approximately 4-fold. Together, these data suggest that the CD40/CD40L is essential for optimal effector function of monocytes, that CD40L-activated monocytes stimulate MC through both soluble factors and cell-to-cell contact mediated pathways, and that both pathways are essential for maximum stimulation of MC.     

C5a suppresses the production of IL-12 by IFN-gamma-primed and lipopolysaccharide-challenged human monocytes.

IL-12 is a key mediator of the immune response, skewing T lymphocytes toward a type 1 cytokine pattern. Priming with IFN-gamma or GM-CSF is required for expression of IL-12p70 by cells in which IL-12 is inducible by bacterial products such as LPS. We here show for the first time that the production of bioactive IL-12 by human monocytes can be significantly suppressed by C5a if applied to IFN-gamma-primed monocytes before LPS stimulation. There was a dose-dependent suppression by IL-12 (p70) on the levels of intracellular cytokine production and cytokine secretion. mRNA studies consistently showed a reduction of IL-12p40 and IL-12p35 expression by stimulation in the presence of C5a. The results of several different experimental approaches suggest that IL-12 down-regulation was not due to endogenous IL-10, IL-4, or PGE2 production induced by C5a. Moreover, stimulation of IFN-gamma-primed monocytes with C5a did not lead to a down-regulation of the CD14 Ag, which is an LPS receptor. These findings show that the anaphylatoxin C5a has the capacity to directly interact with the complex regulation of IL-12.     

Macrophage inflammatory protein-3 beta enhances IL-10 production by activated human peripheral blood monocytes and T cells.

We report that the addition of human macrophage inflammatory protein-3 beta (MIP-3 beta) to cultures of human PBMCs that have been activated with LPS or PHA results in a significant enhancement of IL-10 production. This effect was concentration-dependent, with optimal MIP-3 beta concentrations inducing more than a 5-fold induction of IL-10 from LPS-stimulated PBMCs and a 2- to 3-fold induction of IL-10 from PHA-stimulated PBMCs. In contrast, no significant effect on IL-10 production was observed when 6Ckine, the other reported ligand for human CCR7, or other CC chemokines such as monocyte chemoattractant protein-1, RANTES, MIP-1 alpha, and MIP-1 beta were added to LPS- or PHA-stimulated PBMCs. Similar results were observed using activated purified human peripheral blood monocytes or T cells. Addition of MIP-3 beta to nonactivated PBMCs had no effect on cytokine production. Enhancement of IL-10 production by MIP-3beta correlated with the inhibition of IL-12 p40 and TNF-alpha production by monocytes and with the impairment of IFN-gamma production by T cells, which was reversed by addition of anti-IL-10 Abs to the cultures. The ability of MIP-3 beta to augment IL-10 production correlated with CCR7 mRNA expression and stimulation of intracellular calcium mobilization in both monocytes and T cells. These data indicate that MIP-3 beta acts directly on human monocytes and T cells and suggest that this chemokine is unique among ligands binding to CC receptors due to its ability to modulate inflammatory activity via the enhanced production of the anti-inflammatory cytokine IL-10.     

Chemokine and cytokine production in susceptible C3H/HeN mice and resistant BALB/c mice during Orientia tsutsugamushi infection

To understand the pathogenesis of scrub typhus, we examined chemokine and cytokine production in susceptible (C3H/HeN) and resistant (BALB/c) mice after infection with O. tsutsugamushi Gilliam. C3H/HeN mice produced high levels of chemokines macrophage inflammatory proteins 1 alpha (MIP-1 alpha ), MIP-2, monocyte chemoattractant protein 1 (MCP-1), and cytokines gamma-interferon (IFN-gamma ), interleukin-12 (IL-12), IL-10, and tumor necrosis factor alpha (TNF-alpha ) in response to O. tsutsugamushi infection, compared to BALB/c mice. Chemokine profiles in infected mice correlated well with the kinetics of inflammatory cell infiltration. Hyperproduction of chemokines and cytokines was observed in another susceptible-infection model (BALB/c-Karp). These results suggest that hyperproduction of chemokines and cytokines are associated with susceptibility during O. tsutsugamushi infection.     

Autoregulation of human monocyte-derived dendritic cell maturation and IL-12 production by cyclooxygenase-2-mediated prostanoid production

PG added to cell culture profoundly affect the in vitro maturation and function of monocyte-derived dendritic cells (MDC). Because unstimulated monocytes express cyclooxygenase (COX)-1, and COX-2 when activated, we examined whether MDC express these enzymes and produce prostanoids that autoregulate maturation and IL-12 production. Immature MDC (I-MDC) and mature MDC express COX-1, but, unlike monocytes, both MDC populations constitutively express COX-2. However, COX-2 regulation in both MDC populations differs from monocytes, as IL-4 does not suppress enzyme expression. COX-2 is functional in MDC as a specific inhibitor, NS-398, significantly reduces PGE(2) production. I-MDC undergoing maturation with soluble CD40 ligand (sCD40L) increase PGE(2) synthesis, but prostanoid synthesis is switched to COX-1. However, with IFN-gamma present, sCD40L-stimulated PG metabolism is redirected to COX-2, and PGE(2) synthesis increases severalfold. Endogenous PG production by MDC does not regulate CD40, CD80, CD86, or HLA DR expression; however, it does promote MDC maturation, as NS-398 significantly reduces CD83 expression in I-MDC matured with sCD40L/IFN-gamma. PG produced through COX-2 also autoregulate IL-12, but the effects are dependent on the MDC maturation state. Blocking COX-2 reduces I-MDC secretion of IL-12p40, whereas it increases IL-12p40 and p70 production by maturing MDC. COX-2-mediated PG production impacts MDC function as maturing these cells in the presence of NS-398 yields MDC that stimulate significantly more IFN-gamma in an allogeneic mixed lymphocyte response than MDC matured without this inhibitor. These studies demonstrate that MDC express both COX isoforms constitutively and produce prostanoids, which autoregulate their maturation and function.     

T cell-independent, TLR-induced IL-12p70 production in primary human monocytes

IL-12p70 is a key cytokine for the induction of Th1 immune responses. IL-12p70 production in myeloid cells is thought to be strictly controlled by T cell help. In this work we demonstrate that primary human monocytes can produce IL-12p70 in the absence of T cell help. We show that human monocytes express TLR4 and TLR8 but lack TLR3 and TLR7 even after preincubation with type I IFN. Simultaneous stimulation of TLR4 and TLR8 induced IL-12p70 in primary human monocytes. IL-12p70 production in peripheral blood myeloid dendritic cells required combined stimulation of TLR7/8 ligands together with TLR4 or with TLR3 ligands. In the presence of T cell-derived IL-4, but not IFN-gamma, stimulation with TLR7/8 ligands was sufficient to stimulate IL-12p70 production. In monocytes, type I IFN was required but not sufficient to costimulate IL-12p70 induction by TLR8 ligation. Furthermore, TLR8 ligation inhibited LPS-induced IL-10 in monocytes, and LPS alone gained the ability to stimulate IL-12p70 in monocytes when the IL-10 receptor was blocked. Together, these results demonstrate that monocytes are licensed to synthesize IL-12p70 through type I IFN provided via the Toll/IL-1R domain-containing adaptor inducing IFN-beta pathway and the inhibition of IL-10, both provided by combined stimulation with TLR4 and TLR8 ligands, triggering a potent Th1 response before T cell help is established.     

Interleukin-6 and transforming growth factor-beta regulate the expression of monocyte chemoattractant protein-1 and colony-stimulating factors in human thyroid follicular cells.

Monocytes and T-lymphocytes, both of which play a pivotal role in immune/inflammatory responses, can be attracted from the circulation into tissues by monocyte chemoattractant protein-1 (MCP-1), and monocytes can be further activated by colony-stimulating factors (CSFs), granulocyte/macrophage CSF (GM-CSF) or macrophage CSF (M-CSF). We examined whether either interleukin-6 (IL-6) or transforming growth factor-beta (TGF-beta), both of which are produced by thyroid follicular cells (TFC), can regulate the production of MCP-1 or CSF(s) in human TFC. IL-6, being effective only in the presence of soluble IL-6 receptor (sIL-6R), stimulated the expression of both MCP-1 and M-CSF, but was inhibitory on GM-CSF expression. On the other hand, TGF-beta stimulated the expression of both MCP-I and GM-CSF, but suppressed M-CSF expression. These results suggest a possible role of IL-6 or TGF-beta on the initiation and/or modulation of thyroid immune/inflammatory responses via MCP-1 production and differential production of GM-CSF or M-CSF by TFC.     

IL-18 induces monocyte chemotactic protein-1 production in macrophages through the phosphatidylinositol 3-kinase/Akt and MEK/ERK1/2 pathways

Macrophages are activated during an inflammatory response and produce multiple inflammatory cytokines. IL-18 is one of the most important innate cytokines produced from macrophages in the early stages of the inflammatory immune response. Monocyte chemoattractant protein (MCP-1) is expressed in many inflammatory diseases such as multiple sclerosis and rheumatoid arthritis, and its expression is correlated with the severity of the disease. Both IL-18 and MCP-1 have been shown to be involved in inflammatory immune responses. However, it has been unclear whether IL-18 is involved in the induction of MCP-1. This investigation was initiated to determine whether IL-18 can induce MCP-1 production, and if so, by which signal transduction pathways. We found that IL-18 induced the production of MCP-1 in macrophages, which was IL-12-independent and was not mediated by autocrine cytokines such as IFN-gamma or TNF-alpha. We then examined signal transduction pathways involved in IL-18-induced MCP-1 production. We found that IL-18 did not activate the IkappaB kinase/NF-kappaB pathway, evidenced by no degradation of IkappaBalpha and no translocation of NF-kappaB p65 to the nucleus in IL-18-stimulated macrophages. Instead, IL-18 activated the PI3K/Akt and MEK/ERK1/2 pathways. Inhibition of either of these pathways attenuated MCP-1 production in macrophages, and inhibition of both signaling pathways resulted in the complete inhibition of MCP-1 production. On the basis of these observations, we conclude that IL-18 induces MCP-1 production through the PI3K/Akt and MEK/ERK1/2 pathways in macrophages.     

Thiol antioxidants inhibit the formation of the interleukin-12 heterodimer: a novel mechanism for the inhibition of IL-12 production

IL-12 is a 75 kDa heterodimeric cytokine composed of two disulfide-linked subunits, p35 and p40, which plays an important role in the regulation of the immune response. We tested the hypothesis that thiol antioxidants might interfere with dimerization of the two IL-12 subunits. We thus studied the effect of reduced glutathione (GSH) and N-acetyl-cysteine (NAC) on IL-12 p75 production by human THP-1 cell stimulated with IFN-gamma and Staphylococcus aureus Cowan strain I (SAC), using ELISAs specific for IL-12 p75 or the p40 subunit. NAC and GSH, but not cystine, at concentrations of 5-10 mM inhibited production of IL-12 p75 but not of the p40 subunit. NAC did not inhibit p40 or p35 mRNA expression in dendritic cells or THP-1 cells, or NF-kappa B activation in THP-1 cells. The effect of NAC was specific for IL-12 p75, as NAC did not affect induction of MHC class II expression by IFN-gamma-stimulated THP-1 cells. IL-12 dimer formation appears to be reduced by NAC also in vivo, because pretreatment with NAC (1 g/kg, orally), before LPS injection in mice, inhibited peak IL-12 p75 serum levels without affecting those of p40. We conclude that thiol levels regulate IL-12 p75 production and that assembly of the heterodimer is a step that might represent a target for pharmacological intervention.     

Modulatory effect of HHV-6 on MCP-1 production by human monocytes

Chemokines represent a large family of proinflammatory proteins that orchestrate leukocyte trafficking to sites of viral infection. Human Herpes virus type 6 (HHV-6) is a typical immunosuppressive agent, as suggested by its tropism. In this study the production of monocyte chemoattractant protein-1 (MCP-1) and interleukin-10 (IL-10) by human peripheral blood monocytes was evaluated during HHV-6 infection. Our results demonstrate that HHV-6 infection triggers monocytes to release MCP-1 and IL-10. The addition of exogenous recombinant MCP-1 upregulates the release of extracellular virus, whereas does not influence the percentage of viral-antigen positive cells. Furthermore, the addition of monoclonal antibodies anti-IL-10 down-regulates MCP-1 release induced by HHV-6. These findings indicate that IL-10 and MCP-1 production was closely related and that the marked amounts of MCP-1 were supported not only by virus but also by virus-induced IL-10.     

Deficient IL-12(p35) gene expression by dendritic cells derived from neonatal monocytes

To gain insight into the defects responsible for impaired Th1 responses in human newborns, we analyzed the production of cytokines by dendritic cells (DC) derived from cord blood monocytes. We observed that neonatal DC generated from adherent cord blood mononuclear cells cultured for 6 days in the presence of IL-4 and GM-CSF show a phenotype similar to adult DC generated from adherent PBMC, although they express lower levels of HLA-DR, CD80, and CD40. Measurement of cytokine levels produced by neonatal DC upon stimulation by LPS, CD40 ligation, or poly(I:C) indicated a selective defect in the synthesis of IL-12. Determination of IL-12(p40) and IL-12(p35) mRNA levels by real-time RT-PCR revealed that IL-12(p35) gene expression is highly repressed in stimulated neonatal DC whereas their IL-12(p40) gene expression is not altered. The addition of rIFN-gamma to LPS-stimulated newborn DC restored their expression of IL-12(p35) and their synthesis of IL-12 (p70) up to adult levels. Moreover, we observed that neonatal DC are less efficient than adult DC to induce IFN-gamma production by allogenic adult CD4(+) T cells. This defect was corrected by the addition of rIL-12. We conclude that neonatal DC are characterized by a severe defect in IL-12(p35) gene expression which is responsible for an impaired ability to elicit IFN-gamma production by T cells.     

Cellular basis of the role of diesel exhaust particles in inducing Th2-dominant response

There is growing evidence that diesel exhaust particles (DEP) can induce allergic diseases with increased IgE production and preferential activation of Th2 cells. To clarify the cellular basis of the role of DEP in the induction of Th2-dominant responses, we examined the effects of DEP on the cytokine production by T cells stimulated with anti-CD3/CD28 Ab and on that by monocyte-derived dendritic cells (MoDCs) stimulated with CD40L and/or IFN-gamma. We examined IFN-gamma, IL-4, IL-5, IL-8, and IL-10 produced by T cells and TNF-alpha, IL-1beta, IL-10, and IL-12 produced by MoDCs using real-time PCR analysis or by ELISA. To highlight the effects of DEP, we compared the effects of DEP with those of dexamethasone (DEX) and cyclosporin A (CyA). DEP significantly suppressed IFN-gamma mRNA expression and protein production, while it did not affect IL-4 or IL-5 mRNA expression or protein production. The suppressive effect on IFN-gamma mRNA expression was more potent than that of DEX and comparable at 30 mug/ml with 10(-7) M CyA. The suppressive effect on IFN-gamma production was also more potent than that of either DEX or CyA. DEP suppressed IL-12p40 and IL-12p35 mRNA expression and IL-12p40 and IL-12p70 production by MoDCs, while it augmented IL-1beta mRNA expression. Finally, by using a thiol antioxidant, N-acetyl cysteine, we found that the suppression of IFN-gamma production by DEP-treated T cells was mediated by oxidative stress. These data revealed a unique characteristic of DEP, namely that they induce a Th2 cytokine milieu in both T cells and dendritic cells.     

Alveolar macrophage-derived cytokines induce monocyte chemoattractant protein-1 expression from human pulmonary type II-like epithelial cells

Many acute and chronic lung diseases are characterized by the presence of increased numbers of activated macrophages. These macrophages are derived predominantly from newly recruited peripheral blood monocytes and may play a role in the amplification and perpetuation of an initial lung insult. The process of inflammatory cell recruitment is poorly understood, although the expression of inflammatory cell-specific chemoattractants and subsequent generation of chemotactic gradients is likely involved. Although immune cells such as macrophages and lymphocytes are known to generate several inflammatory cell chemoattractants, parenchymal cells can also synthesize and secrete a number of bioactive factors. We now demonstrate the generation of significant monocyte chemotactic activity from tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 beta-treated pulmonary type II-like epithelial cells (A549). The predominant inducible monocyte chemotaxin had an estimated molecular mass of approximately 14-15 kDa and was neutralized by specific antibody to human monocyte chemotactic protein-1 (MCP-1). Induction of activity was accompanied by increases in steady-state mRNA level for MCP-1. These data are consistent with the induction of MCP-1 expression from A549 cells by TNF and IL-1. MCP-1 production from A549 cells could be induced by lipopolysaccharide (LPS)-stimulated alveolar macrophage (AM)-conditioned media, but not by LPS alone. The inducing activity in AM-conditioned media was neutralized with specific antibodies to IL-1 beta, but not TNF-alpha. Our findings suggest that the alveolar epithelium can participate in inflammatory cell recruitment via the production of MCP-1 and that cytokine networking between contiguous alveolar macrophages and the pulmonary epithelium may be essential for parenchymal cell MCP-1 expression.     

Flow cytometric analysis of cytokine production by normal human peripheral blood dendritic cells and monocytes: comparative analysis of different stimuli, secretion-blocking agents and incubation periods

In this paper, we comparatively analyze the effects of the following different stimuli on the production and intracellular accumulation of the interleukin (IL)-1 beta, IL-6, IL-12, tumor necrosis factor-alpha (TNF-alpha), and IL-8 inflammatory cytokines in both normal human peripheral blood (PB) dendritic cell (DC) subsets and monocytes: lipopolysaccharide (LPS) versus Staphylococcus aureus cowan I (SAC) in the presence or absence of interferon-(IFN)-gamma-, cytokine secretion-blocking agents (brefeldin A alone versus brefeldin A plus monensin), and incubation periods (6, 12, and 24 h). For this purpose, a four-color multiple-staining direct immunofluorescence technique analyzed by flow cytometry was systematically used in all experiments (n = 19). Our results show that after stimulation, an important proportion of each of the two CD33(+) myeloid DC subsets as well as the monocytes produce significant amounts of all cytokines analyzed under each of the experimental conditions assayed. In contrast, CD33(-/+lo) lymphoplasmocytoid DC failed to produce detectable levels of any of the above-mentioned cytokines under the same stimulatory conditions. Upon comparing the different stimuli used, LPS was associated with higher percentages of cytokine-producing cells compared with SAC, especially within the CD33(hi) DC subset; interestingly, the addition of IFN-gamma enhanced the response of monocytes to both LPS and SAC. As regards the secretion-blocking agents, brefeldin A alone was superior to the combination of brefeldin A and monensin. This is because it was frequently associated with both a higher percentage of cytokine-positive cells and greater amounts of detectable cytokines per cell. Sequential analysis of cytokine production by PB DC and monocytes after 6, 12, and 24 h of cell culture showed that after 6 h, an increased cell death rate existed among DC, which became even undetectable at 24 h, in the absence of a significant increase in cytokine secretion. In summary, our results show that from the experimental conditions assayed in this paper, to induce cytokine production by normal human DC and monocytes, maximum response is obtained once PB samples are stimulated for 6 h with LPS (with or without IFN-gamma) in the presence of brefeldin A alone.     

Functional and structural regression of the rabbit corpus luteum is associated with altered luteal immune cell phenotypes and cytokine expression patterns

Following attenuation of progesterone production corpora lutea are selectively cleared, a process associated with recruitment of macrophages. In the rabbit little is known about luteal immune cell phenotypes and expression of cytokines, which influence immune cells and resident luteal cells, during luteolysis. Consequently, we studied luteal immune cells by immunohistochemistry as well as luteal IL-10, TNFalpha, MCP-1, IFN-gamma, and IL-1beta mRNA expression by semiquantitative RT-PCR from day 8 to day 20 in pseudopregnant rabbits (d8-d20 p.hCG). Luteal function was assayed by serum progesterone levels. Functional luteolysis commenced by d14 p.hCG as indicated by attenuation of serum progesterone levels. X4(+) tissue macrophage levels increased transiently on d12 and d14 p.hCG, whereas CD5(+) T-cell levels transiently declined on these two days. CD68(+) macrophages increased progressively after d16 p.hCG. The luteal mRNA level of the anti-inflammatory cytokine IL-10 as well as the mRNA levels of the pro-inflammatory cytokines TNFalpha and MCP-1 increased after d16 p.hCG and remained elevated up to d20 p.hCG. IFN-gamma and IL-1beta mRNA expression did not vary systematically. In summary, luteolysis was associated with an initial transient increase of X4(+) macrophages and decrease of CD5(+) T-cells, and later recruitment of CD68(+) macrophages. During structural regression pro- and anti-inflammatory cytokines are upregulated possibly to control immune cell function.