Regulation of Toll-like receptor (TLR)2 and TLR4 on CD14dimCD16+ monocytes in response to sepsis-related antigens

Rapid overproduction of proinflammatory cytokines are characteristic of sepsis. CD14(dim)CD16(+) monocytes are thought to be major producers of cytokine and have been shown to be elevated in septic patients. Toll-like receptors (TLR) are pattern recognition receptors important in mediating the innate immune response and their activation can lead to production of cytokines. Using whole blood culture and flow cytometry we have investigated TLR2 and TLR4 regulation after stimulation with sepsis-relevant antigens [lipopolysaccharide (LPS), Staphylococcal enterotoxin B (SEB) and peptidoglycan (PGN)]. The percentage of CD14(dim)CD16(+) monocyte population expanded at 20 h post-stimulation, after a rise in tumour necrosis factor (TNF)-alpha and interleukin (IL)-6 at 2 h. A strong positive correlation between the percentage of CD14(dim)CD16(+) monocytes and secreted TNF-alpha was demonstrated (r = 0.72). Furthermore, we were able to induce expansion of the CD14(dim)CD16(+) population to approximately 35% of all monocytes with the addition of recombinant TNF-alpha to the whole blood culture. TLR4 was found to be expressed 2.5 times higher on CD14(dim)CD16(+) compared to CD14(+) CD16(-) monocytes, while TLR2 expression was similar in both subpopulations. The CD14(dim)CD16(+) and CD14(+) CD16(-) monocyte populations were different in their response to various antigens. LPS down-regulated TLR4 by 4.9 times in CD16(+) monocytes compared to only 2.3 times in CD16(-) monocytes at 2 h. LPS was able to up-regulate TLR2 by 6.2 times after 2 h, with no difference between the subpopulations. LPS further up-regulated TLR2 by 18.4 times after 20 h only in the CD14(+) CD16(-) population. PGN and SEB induced no significant changes in TLR2 or TLR4 expression. We hypothesize that following exposure to bacterial antigens, subsequent TNF-alpha drives a differentiation of monocytes into a CD14(dim)CD16(+) subpopulation.     

Colony-stimulating factors and interferon-gamma differentially affect cell surface molecules shared by monocytes and neutrophils

Monocytes and neutrophils share a common progenitor and perform many similar functions, as reflected in the expression of several shared membrane molecules. We show here that such molecules can be independently regulated by the cytokines interferon-gamma (IFN-gamma) and the colony-stimulating factors (CSF) in the context of the cell type on which they are present. Thus, IFN-gamma causes neutrophils to express the high-affinity receptor for IgG, Fc gamma RI, which has been considered to be a monocyte-specific receptor. Although neutrophil Fc gamma RI never reaches the levels present on monocytes, it is induced more rapidly and by lower amounts of IFN-gamma than monocyte Fc gamma RI. Of the CSF, only macrophage (M) CSF has an effect which is to cause a decrease in expression of monocyte Fc gamma RI. Secondly, after culture monocytes express Fc gamma RIII which is constitutively expressed by neutrophils. Although neutrophil Fc gamma RIII can be readily modulated by IFN-gamma, granulocyte (G) CSF and granulocyte-macrophage (GM) CSF, these cytokines do not alter the low levels of monocyte Fc gamma RIII. Thirdly, expression of the gp55 protein recognized by CD14 monoclonal antibodies is decreased after exposure of monocytes to IFN-gamma. Neutrophils express low levels of CD14 and, in this case, IFN-gamma causes an increase in the CD14 antigen on these cells. Of all the molecules investigated, only HLA class II is confined to monocytes, with increases in expression induced by IFN-gamma but not by the CSF.     

Triggering of respiratory burst by phagocytosis in monocytes of patients with systemic lupus erythematosus.

The triggering of the respiratory burst by phagocytosis via different receptors in monocytes of patients with systemic lupus erythematosus (SLE) was investigated. The superoxide anion synthesis was assayed by reduction of ferricytochrome C that was inhibited by superoxide dismutase. The mononuclear cell suspensions were triggered by IgG-coated latex, C3 complement fragment-coated and uncoated yeast (Saccharomyces cerevisiae). Superoxide generation induced by phagocytosis via Fc gamma R was decreased in monocytes of patients with SLE. On the other hand, MoAbs against Fc gamma RI, Fc gamma RII and especially CR3 could also induce superoxide anion synthesis. At the same time, superoxide generation induced by anti-CR3 could be inhibited with C3-coated yeast.     

Measurement of soluble Fcgamma receptor type IIIa derived from macrophages in plasma: increase in patients with rheumatoid arthritis

FcgammaRIII (CD16) is found in two alternative forms, a transmembrane FcgammaRIIIa expressed on NK cells and macrophages, and a glycosylphosphatidylinositol-linked FcgammaRIIIb present on neutrophils. Previously, we measured soluble FcgammaRIIIa (sFcgammaRIIIa) in plasma of NA(1 +, 2-) phenotyped donors with the anti-FcgammaRIII monoclonal antibody (MoAb) GRM1, which recognizes NA2-FcgammaRIIIb and FcgammaRIIIa. The level of sFcgammaRIIIa, as well as the total sFcgammaRIII (sFcgammaRIIIa plus sFcgammaRIIIb) in patients with rheumatoid arthritis (RA) was significantly higher than that in healthy controls. In this study, we measured sFcgammaRIIIa(M)(phi) in plasma with a newly developed anti-FcgammaRIII MoAb, MKGR14 (mIgM), which recognizes FcgammaRIIIa(M)(phi) specifically. From the recovery of purified sFcgammaRIIIa(M)(phi), the amount of sFcgammaRIIIa(M)(phi) present was about half that of sFcgammaRIIIa(NK), and that of sFcgammaRIIIa was about 50 times lower than that of sFcgammaRIIIb in pooled plasma from healthy NA(1 +, 2-) phenotyped donors. The level of sFcgammaRIIIa(M)(phi) in RA patients was about four times higher than that in healthy controls. In RA patients, both the sFcgammaRIIIa(M)(phi) and sFcgammaRIIIa levels were increased as proportionally as the Lansbury Index. The sFcgammaRIIIa, but not sFcgammaRIIIa(M)(phi) levels, were increased directly proportional to C-reactive protein. sFcgammaRIIIa(M)(phi) may be a novel marker of disease activity in RA.     

BOOSTING T CELL COSTIMULATION IN CANCER: THE POSSIBILITIES SEEM ENDLESS

Effective immune strategies for eradication of human malignancies will require a thorough understanding of the interactions of cancer with the immune system. It will be crucial to understand how to optimize and sustain a T cell immune response. Recently, our understanding of the molecular interaction that occurs between an APC and a T cell during cognate interaction has increased dramatically. In this review, various costimulatory and inhibitory molecules of the B7 and TNF families will be discussed. The emphasis will be on how these costimulatory molecules impact T cell activation and on how they can be potentially used for the treatment of cancer. costimulation cancer T cell activation     

Increased expression of IgG Fc receptor type I on neutrophils and monocytes from HIV-infected subjects.

Interferon-gamma (IFN-gamma) induces de novo expression of IgG Fc receptor type I (FcRI) on neutrophils and significantly raises the level of these receptors on monocytes. Since increased concentrations of IFN-gamma have been observed in sera from patients with HIV infection, FcRI expression might also be increased on these subjects' phagocytes. FcRI expression was assessed by indirect immunofluorescence staining of phagocytes in whole blood from 40 healthy controls and 55 HIV+ subjects, 24 belonging to CDC class III and 31 to CDC class IV; 42 were intravenous drug abusers (IVDA) and 13 were homosexual men. Plasma levels of IFN-gamma were measured using a modified immunoradiometric assay. The mean linear fluorescence intensity, used as a relative measure of receptor expression, was significantly higher on unseparated neutrophils from HIV+ subjects in CDC classes III (P < 0.001) and IV (P < 0.0001) than from controls. Similar changes in FcRI expression were observed on monocytes from HIV+ subjects. While no differences were observed between IVDA and homosexual HIV+ patients, there was a significant association between FcRI expression and the patients' CDC stage, those in class IV having the highest FcRI levels. Plasma IFN-gamma concentrations were significantly higher in HIV+ patients than in controls and a positive correlation with the stages of HIV infection was again observed. FcRI expression was also increased on freshly purified neutrophils from five HIV+ patients in CDC class IV but did not increase further after 18 h incubation with IFN-gamma, a treatment that up-regulated FcRI expression on control neutrophils. These data suggest that: (i) FcRI evaluation may be a sensitive marker for the biological activity of IFN-gamma in vivo; (ii) phagocytes from HIV+ subjects are activated in vivo by IFN-gamma, expressing increased levels of FcRI; (iii) these IFN-gamma-activated cells may play a role in the pathogenesis of AIDS.     

Activation-induced proteolysis of the cytoplasmic domain of ζ in T cell receptors and Fc receptors

The CD3-T cell receptor (TCR) complex on T cells and the Fcγ receptor type III (FcγRIII)-ζ-γ complex on natural killer cells are functionally analogous activation receptors that associate with a family of disulfide-linked dimers composed of the related subunits ζ and γ. Immunochemical analysis of receptor complexes separated on two-dimensional diagonal gels allowed the identification of a previously uncharacterized ζ-p14 heterodimer. ζ-p14 is a component of both CD3-TCR and FcγRIII-ζ-γ. Peptide mapping analysis shows that p14 is structurally related to ζ, suggesting that it is either: (i) derived from ζ proteolytically or (ii) the product of an alternatively spliced mRNA. The observation that COS cells transformed with a cDNA encoding ζ express ζ-p14 supports the former possibility. The expression of CD3-TCR complexes including ζ-p14 increases following activation with phorbol 12-myristate 13-acetate or concanavalin A, suggesting that proteolysis of ζ may contribute to receptor modulation or desensitization.     

Production by cultured human monocytes of mesangial cell proliferation factor(s) differing from interleukin-1 and interleukin-6.

Conditioned media from human peripheral blood leucocytes treated with lipopolysaccharide (LPS) induced a marked increase in the 3H-thymidine incorporation of cultured mesangial cells at low serum concentration (four to six times higher than control). Two sizes (100-70 and 8-12 kD) of monocyte-derived mesangial cell proliferating factors (MDF) were separated by column chromatography. Their peaks were distinct from those of thymocyte proliferating activity. The addition of anti-human interleukin-1 (IL-1) or anti-recombinant human interleukin-6 (IL-6) antibody to the fractionated MDF failed to have any effect on the mitogenic activity toward mesangial cells. The addition of anti-human platelet-derived growth factor (PDGF) antibody to the low molecular weight fraction decreased mesangial cell mitogenic activity (40-60% of control), but addition to the higher fraction did not (80-100% of control). From these data it seems that a large portion of the monocyte-derived mesangial cell growth factor was not comprised of IL-1 or IL-6 but of PDGF-like molecules; and that there is an unknown mesangial cell proliferating factor (or factors) besides IL-1, IL-6 and PDGF.     

Up-regulation of tumour necrosis factor-alpha receptors on monocytes by desferrioxamine.

The effect of endogenously generated reactive oxygen metabolites on the interaction of human blood monocytes with tumour necrosis factor-alpha (TNF-alpha) was investigated. Pre-exposure of unactivated human blood monocytes to dimethylthiourea, a scavenger of hydroxyl radical (OH.), or to desferrioxamine (DFX), an iron chelator preventing the synthesis of OH., enhanced the specific binding of 125I-TNF-alpha to its receptors. Scavengers of superoxide anion or hydrogen peroxide were without effect. DFX-induced up-regulation of 125I-TNF-alpha binding depended on the concentration of the drug (1-5 mM) and on the duration of the treatment (1-18 h). It was not due to a reduction of receptor occupancy by endogenously generated TNF-alpha. Scatchard analysis of binding data revealed that DFX caused an approximately two-fold increase in the number of type II TNF-alpha receptors, with no change in their affinity. This up-regulation, that did not require synthesis of new proteins, was associated with a decrease in the internalization rate of TNF-alpha receptors, the half-life of which was doubled. Conversely, these findings suggest that OH. generation by monocytes may have a physiological role in reducing the activity of membrane-associated TNF-alpha receptors.     

Plasmacytoid monocytes (so-called plasmacytoid T cells) in Hodgkin's disease

The occurrence and distribution of plasmacytoid monocytes (so-called plasmacytoid T cells) were investigated immunohistochemically in 40 cases of Hodgkin's disease. Large numbers of plasmacytoid monocytes were found in all cases of lymphocyte predominance, nodular sclerosing, and mixed cellularity Hodgkin's disease, characterized by a minor degree of architectural effacement. They occurred at the periphery of lymphoid aggregates which mimic the composite nodule of the reactive lymph node and which contained Reed-Sternberg cells and their variants. Despite some immunophenotypic similarities, no further arguments were found to support a relationship between plasmacytoid monocytes and Reed-Sternberg cells. We conclude that plasmacytoid monocytes represent one of the monocyte-derived cells that contribute to the cellular reaction in Hodgkin's disease.     

FcγR Ⅱ a基因多态性、中性粒细胞胞浆抗体、循环免疫复合物在系统性红斑狼疮发病肿饔玫难芯靠

目的 通过检测系统性红斑狼疮(SLE)病人FcγR Ⅱ a受体的基因型及其分布情况,检测了血清中的中性粒细胞胞浆抗体(ANCA)、循环免疫复合物(CIC).分析三者之间的关系和其与SLE的关系,从基因表达、转录及表达产物的作用三个层次探讨了SLE的发病机制.方法 收集69例SLE病人与48例健康人的标本,采用巢式PCR对FcγRⅡa基因进行分型,RT-PCR检测该基因的转录,采用ELISA方法对血清中的ANCA和CIC进行检测.结果 FcγR Ⅱ a-R131纯和子的分布在SLE患者组与正常对照组之间有统计学意义(P<0.02).三种基因型在狼疮病人中(分别为45%Fcγ/RⅡa-R/R131、30%H/R 131和25%H/H131)与正常对照组(21%FcγR Ⅱ a-R/R131、52%H/R131和27%H/H131)之间不同.FcγRⅡ a-R/R131基因型转录产物在SLE患者组与正常对照组之间有有统计学意义(P<0.02),此结果与分型结果一致.在FcγR Ⅱ a-R/R131基因型中抗ANCA阳性率为67.9%,CIC阳性率67.4%,与FcγRⅡ a-H/R131和FcγRⅡ a-H/H131两个基因型(ANCA:21.4%H/R131和10.7%H/H131;CIC:18.4%H/R131;14.0%H/H131)之间有统计学意义(P<0.05).ANCA与CIC的阳性率在H/R131与H/H131基因型之间无统计学意义(P0.2).结论 FcγR Ⅱ a-R/R基因型可能是云南汉族SLE病人的遗传易感因素,其在体内的过度表达可能是造成SLE患者血清中ANCA和CIC含量增高进而导致SLE发生的一个重要原因.     

Effects of human placental lactogen on the expression of CD163 and CD14 on human monocytes in culture

The effect of human placental lactogen (hPL), a member of the somatomammotrophin family, on the regulation of the scavenger receptor molecules CD14 and CD163 on human monocytes cultured for 48h was investigated. Cells were cultured in the presence or absence of the hormone and also in the presence or absence of IFN-gamma and dexamethasone. Monocytes cultured in the presence of hPL showed a significant increase in the expression of CD14 in both males and females compared to background. When IFN-gamma and dexamethasone were added to the cultures, CD14 expression was decreased and was not rescued by the presence of hPL. hPL alone had no effect on the expression of CD163 on cultured monocytes from either gender, although cells cultured in the presence of IFN-gamma and dexamethasone showed a profound increase in their expression of CD163. This expression was augmented further by the presence of hPL in the cultures over a 48-h period. These results support the hypothesis of a potential role of this hormone in the regulation of the innate immune response.     

Characterization of biological response modifier release by human monocytes cultured in suspension in serum-free medium

Human monocytes have been shown to be critical immunoregulatory cells for a variety of frequently measured in vitro human immune functions and are secretors of potent biologic response modifiers (BRMs). These BRMs, such as interferon (IFN), colony stimulating factor (CSF) and prostaglandin E (PGE), may play important roles in the host immune response to cancer. A new approach for culturing circulating peripheral blood monocytes has been developed to retain the native characteristics of these cells, avoiding possible alteration or activation by adherence. The ability of elutriator-purified human monocytes to secrete IFN and PGE was examined under conditions of suspension culture as well as after adherence, and no difference in secretion of these BRMs was noted. In contrast, Teflon-cultured monocytes demonstrated a significantly enhanced CSF release over culturing in polystyrene plates. A new serum-free medium has also been developed, and monocytes cultured in vitro in this medium showed a 5-fold increase in IFN release, and up to a 72% increase in CSF release when compared to optimal standard culture medium (containing 10% AB serum) and an increased stimulation index for PGE release. By these procedures, hundreds of millions of highly purified human monocytes can be sterilely isolated in suspension and cultured in suspension in serum-free medium with retention of BRM-releasing capabilities. This system should permit more detailed molecular studies of monocytes (in which serum can be an impediment) and may also facilitate clinical therapy studies involving the in vivo transfer of monocytes activated in suspension in vitro.     

Macrophage T-cell interaction in man: Handling of tetanus toxoid antigen by human monocytes

An absolute requirement for monocytes was demonstrated in the T-cell proliferative response to tetanus toxoid (TT) antigen. Antigen-pulsed monocytes were shown to be effective in triggering T-cell proliferation. Using¹²⁵I-radiolabeled TT antigen, uptake by monocytes increased progressively over an 18-hr period, at which time 80–85% of the monocytes contained radiolabeled material. The ability of antigen-pulsed monocytes to trigger T-cell proliferation paralleled antigen uptake over an 18-hr period. Monocytes pulsed with antigen, then washed, lost their ability to trigger T-cell proliferation following a 24- to 48-hr culture period. Metabolic inhibitors blocked antigen uptake by monocytes and monocyte triggering of T-cell proliferation. Trypsin treatment of TT-pulsed monocytes did not affect the amount of antigen associated with monocytes or T-cell triggering by monocytes. Anti HLADR alloantibodies, which when added during antigen pulsing of monocytes inhibit the capacity of these monocytes to trigger T-cell proliferation, did not interfere with antigen uptake. These results indicate that human monocytes present antigen to T cells via an active process and in association with DR determinants, and that the immunogenic moiety of antigen does not remain indefinitely available to the T cell.     

Increased expression and occupancy of receptors for tumour necrosis factor on blood monocytes from tuberculosis patients.

Blood monocytes from tuberculosis patients release high amounts of tumour necrosis factor-alpha (TNF-alpha). Because the biological efficiency of TNF-alpha would depend on the expression of TNF-alpha receptors on target cells, we thought to analyse the capacity of blood monocytes from a group of patients with pulmonary tuberculosis to bind 125I-TNF-alpha. We report a slight but not significant enhancement in specific binding of 125I-TNF-alpha on monocytes of 15 consecutively studied patients compared with 10 controls. Per cent cell surface bound and internalized 125I-TNF-alpha was identical in the two groups. To evaluate the receptor occupancy by endogenously generated TNF-alpha, similar experiments were performed after cell exposure to low-pH glycine buffer. Under these conditions, specific binding of 125I-TNF-alpha was significantly higher on tuberculosis monocytes compared with control monocytes. Moreover, the occupancy of TNF-alpha receptors by endogenously generated TNF-alpha that was found to be significantly higher on tuberculosis monocytes than on control monocytes, was directly related to the enhanced capacity of mononuclear cells to generate TNF-alpha in vitro. It normalized after 3 months of antituberculous therapy. Scatchard analysis of the binding data revealed that tuberculosis infection caused a significant increase in high affinity 125I-TNF-alpha binding to monocytes without any significant change in the dissociation constant. Collectively, these results indicate an up-regulation of TNF-alpha generation and binding to blood monocytes in patients with pulmonary tuberculosis. They provide support to the hypothesis that TNF-alpha is of critical importance in the pathogenesis of this infection.     

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has opposing effects on the capacity of monocytes versus monocyte-derived dendritic cells to stimulate the antigen-specific proliferation of a human T cell clone

GM-CSF is widely used in combination with IL-4 to differentiate monocytes into potent T cell stimulatory cells, referred to as monocyte-derived dendritic cells (MoDC). These cytokines further increased the stimulatory function of MoDC when present during their incubation with antigen, as determined by the proliferative response of an allergen-specific T cell clone. Conversely, the incubation of freshly isolated monocytes with antigen in the presence of GM-CSF or GM-CSF and IL-4 strongly inhibited the specific stimulation of the T cells, compared with monocytes pulsed in the absence of cytokines. This suppression was partly due to the secretion of prostaglandin E2 (PGE2) and IL-10 by GM-CSF-treated monocytes, since the combined use of indomethacin and anti-IL-10 antibodies during GM-CSF incubation and antigen pulsing restored T cell growth to about 65% of control levels. As confirmed by culture supernatant transfer experiments, maximal inhibition of T cell stimulation was also dependent on the direct contact between the T cells and GM-CSF-treated monocytes during antigen presentation. Collectively, these results imply that GM-CSF can either inhibit or enhance the re-stimulation of primed T cells by antigen-presenting monocytes or MoDC, respectively.     

Differential expression of Toll-like receptor (TLR)-2 and TLR-4 on monocytes in human sepsis

Toll-like receptors (TLRs) are a recently described family of immune receptors involved in the recognition of pathogen-associated molecular patterns (PAMPs). The central role of TLR-2 and TLR-4 in microbial responses suggests they may be implicated in the pathogenesis of human sepsis. We hypothesized that the incidence and outcome of sepsis would be influenced by the expression of TLR-2 and TLR-4 on monocytes. We have examined the expression of TLR-2 and TLR-4 mRNA and protein and their response to pro- and anti-inflammatory agents on monocytes from subjects in the intensive therapy unit (ITU) with and without Gram-negative, Gram-positive or polymicrobial sepsis. We compared these data to ITU and healthy control subjects. TLR-2 mRNA was significantly up-regulated on monocytes from subjects with both Gram-positive and Gram-negative sepsis. Similarly, we detected increased levels of TLR-2 protein on the surface of monocytes from sepsis subjects relative to ITU controls. TLR-4 mRNA was increased in Gram-positive subjects; however, there was no corresponding increase in TLR-4 protein. Although TLR-4 mRNA expression in healthy control monocytes could be modulated in vitro by culture with lipopolysaccharide or interleukin-10, this was not observed in monocytes obtained from sepsis and ITU control subjects, suggesting that septic and ITU control milieus may alter the immunoregulation of TLR-4 mRNA expression on monocytes. TLR-2 mRNA was not modulated in culture by any stimulus in any group. We suggest that expression and regulatory response of monocyte TLR-2, and to a lesser extent TLR-4 may be abnormal in human sepsis.