Heterogeneity of peripheral blood monocytes, endothelial dysfunction and subclinical atherosclerosis in patients with systemic lupus erythematosus.

BACKGROUND: Systemic lupus erythematosus (SLE) is characterized by increased cardiovascular morbidity and mortality. SLE patients have increased prevalence of subclinical atherosclerosis, although the mechanisms of this observation remain unclear. Considering the emerging role of monocytes in atherosclerosis, we aimed to investigate the relationship between subclinical atherosclerosis, endothelial dysfunction and the phenotype of peripheral blood monocytes in SLE patients. METHODS: We characterized the phenotype of monocyte subsets defined by the expression of CD14 and CD16 in 42 patients with SLE and 42 non-SLE controls. Using ultrasonography, intima-media thickness (IMT) of carotid arteries and brachial artery flow-mediated dilation (FMD) as well as nitroglycerin-induced dilation (NMD) were assessed. RESULTS: Patients with SLE had significantly, but only modestly, increased IMT when compared with non-SLE controls (median (25th/75th percentile) 0.65 (0.60/0.71) mm vs 0.60 (0.56/0.68) mm; p < 0.05). Importantly, in spite of early atherosclerotic complications in the studied SLE group, marked endothelial dysfunction was observed. CD14dimCD16+proinflammatory cell subpopulation was positively correlated with IMT in SLE patients. This phenomenon was not observed in control individuals. Interestingly, endothelial dysfunction assessed by FMD was not correlated with any of the studied monocyte subsets. CONCLUSIONS: Our observations suggest that CD14dimCD16+monocytes are associated with subclinical atherosclerosis in SLE, although the mechanism appears to be independent of endothelial dysfunction.

Characterization of the impairment of the uptake of apoptotic polymorphonuclear cells by monocyte subpopulations in systemic lupus erythematosus.

Efficient removal of apoptotic polymorphonuclear leukocytes (PMNs) is an important step in the resolution of inflammation, which protects tissues from the noxious contents of dying cells. While the impairment of apoptotic PMNs removal has been demonstrated for macrophages in systemic lupus erythematosus (SLE), recent studies show that monocytes are also capable of such phagocytosis, although their involvement in SLE is not clear. Therefore, we characterized phagocytosis of apoptotic PMNs by monocytes in 22 patients with SLE and 22 healthy controls. Using flow cytometry we demonstrate that in SLE peripheral blood monocytes show impaired phagocytosis of autologous apoptotic PMNs, while they efficiently engulf apoptotic PMNs isolated from healthy subjects. Monocytes CD14highCD16+ and CD14dimCD16+ more efficiently interacted with apoptotic neutrophils than CD16- cells both in SLE and healthy subjects. Monocytes in SLE showed modestly decreased expression of CD35 and CD91 and increased expression of T Cell Ig- and mucin-domain-containing molecule-3 (TIM-3); however, these differences were evident mainly in selected subsets of monocytes (CD16+) while defects in phagocytosis were observed in all monocyte subsets. Apoptotic cell-dependent induction of lipopolysaccharide (LPS) stimulated production of anti-inflammatory cytokine IL-10 by peripheral blood mononuclear cells (PBMC) was blunted in SLE while the production of pro-inflammatory cytokine TNF-α was unchanged.

Peripheral blood CD14high CD16+ monocytes are main producers of IL-10.

Based on CD14 and CD16 expression, human peripheral blood monocytes (MO) can be divided into a major CD14(high) CD16(-) population and two minor CD14(high) CD16(+) and CD14(dim) CD16(+) subpopulations. CD14(dim) CD16(+) MO are well characterized and regarded as pro-inflammatory because upon stimulation produce TNF-alpha but little, if any, IL-10. By contrast, little is known about CD14(high) CD16(+) MO. We investigated the surface expression of selected determinants by CD16(+) MO subpopulations, cytokine production, phagocytosis and antigen presentation. We found that both CD16(+) subpopulations had a higher expression of HLA-DR, CD86, CD54 and a lower expression of CD64 than CD14(high) CD16(-) population. In addition, CD14(high) CD16(+) MO showed a higher expression of CD11b and TLR4 than CD14(dim) CD16(+) and CD14(high) CD16(-) subpopulations. CD14(high) CD16(+) MO exhibited an increased phagocytic activity and a decreased antigen presentation in comparison with CD14(dim) CD16(+). As expected, lipopolysaccharide (LPS)-stimulated CD14(dim) CD16(+) MO produced TNF-alpha but little IL-10. By contrast, LPS-stimulated CD14(high) CD16(+) subpopulation produced significantly more IL-10 than CD14(dim) CD16(+) and CD14(high) CD16(-) MO. In conclusion, our data show that human peripheral blood CD16(+) MO are heterogeneous in function and consist of two subpopulations: CD14(dim) CD16(+) pro-inflammatory and CD14(high) CD16(+) with anti-inflammatory potential.

Effects of novel plant antioxidants on platelet superoxide production and aggregation in atherosclerosis.

Superoxide anion is produced in human platelets predominantly by Nox2-dependent NADPH oxidases. In vitro experiments have shown that it might play a role in modulating platelet functions. The relationship between platelet superoxide production and aggregation remains poorly defined. Accordingly, we aimed to study superoxide production and aggregation in platelets from subjects with significant cardiovascular risk factors (hypertension, hypercholesterolemia, smoking and diabetes mellitus) and from control individuals. Moreover, we studied the effects of novel polyphenol-rich extracts of Aronia melanocarpa (chokeberry) berries on platelet function in vitro. Superoxide production was significantly increased in patients with cardiovascular risk profile when compared to controls, while platelet aggregation in response to either collagen or thrombin were borderline higher, and did not reach statistical significance. Interestingly, no relationship was observed between platelet aggregation ex vivo and platelet superoxide production in either of studied groups. No correlation was found between endothelial function (measured by FMD) and platelet aggregation ex vivo either. Polyphenol-rich extracts of A. melanocarpa berries caused a significant concentration dependent decrease in superoxide production only in patients with cardiovascular risk factors, while no effect was observed in the control group. A. melanocarpa extracts abolished the difference in superoxide production between risk factor patients and controls. A. melanocarpa extracts exerted significant concentration dependent anti-aggregatory effects in both studied groups, which indicated that these effects may be independent of it's ability to modulate superoxide production. The anti-aggregatory effects of chokeberry extracts were similar irrespective of aggregation inducing agent (collagen or thrombin). Moreover, they appear to be independent of platelet NO release as NOS inhibition by L-NAME did not lead to their abrogation.

Distinct populations of antigen-presenting macrophages are required for induction of effector and regulatory cells in contact sensitivity response in mice.

Macrophages (Mf) and other antigen-presenting cells (APCs) are able to induce both immune and regulatory T cells. We compared the antigen-presenting activities of different subpopulations of thioglycolate-induced peritoneal macrophages from mice that were or were not treated with cyclophosphamide (CY) by several functional (adherence and phagocytosis) and morphologic (phenotypic) markers (FcR, Ia). Different subpopulations of macrophages were derivatized with trinitrophenyl and injected intravenously into recipients, which were tested directly for a contact sensitivity (CS) reaction or for the presence of efferent suppressor T (Ts) cells in passive transfer experiments. Our results demonstrate that peritoneal macrophages are both morphologically and functionally heterogeneous. Macrophages that induce immune cells that mediate CS have characteristics different from those that induce Ts cells. They accumulate in the low-density cell fraction on a discontinuous Ficoll gradient, are insensitive to treatment in vivo with low doses of CY, phagocytose poorly and adhere to plastic, and perhaps have low expression of FcRI and FcRII. Both macrophage fractions seem not to differ in expression of Ia, Mac-1, and Mac-3 antigens. It is argued that low doses of CY abrogate suppression in vivo by selective action on Ts cells. Our results confirm that at least a portion of the action of CY may be due to its influence on certain subpopulations of APCs.

Human T cell subsets differ in their ability to migrate in vitro and to produce T cell migration inhibitory factor.

The spontaneous migration in vitro, production of T cell migration inhibitory factor (TIF) and the response to TIF of OKT4+ and OKT8+ human T cell subsets were studied. The OKT4+ lymphocytes migrated far better than the OKT8+ cells although the movement of both subsets was comparably inhibited by TIF. The OKT8+ subset was found to be a major source of TIF, while OKT4+ cells were responsible for macrophage migration inhibitory factor (MIF) production. The implications of lymphokine production by OKT8+ cells for the regulation of inflammatory responses are discussed.

Immunoregulation of lymphoproliferation in vitro by monocytes and their subpopulations. I. The induction of suppressor T cells in long term cultures and the role of MHC class II determinants and preliminary characteristics.

Peripheral blood monocyte (MO) subpopulations isolated on a basis of functional expression or a lack of Fc receptor (FcR+ and FcR- MO) were used to study the regulation of the antigen (PPD)-driven lymphoproliferation. Long-term cultures of T lymphocytes with FcR+ MO, but not FcR- MO, led to the induction of T suppressor (Ts) cells that inhibited antigen-driven lymphoproliferation in the test cultures. These Ts cells were resistant to mitomycin C, belonged to afferent-acting category of Ts cells, expressed CD8 and HLA-DR determinants, and showed no antigenic specificity nor genetic restriction in action. The expression of MHC class II molecules (HLA-DR and HLA-DP but not HLA-DQ determinants) on MO which were used for antigen presentation was critical for Ts cell induction. It was concluded that specialized MO subpopulations may regulate the lymphoproliferation by inducing Ts cells (or Ts cell circuit) that in turn inhibit antigen-driven immune response.

Pokeweed mitogen activated suppressor T-cells preferentially reduce immunoglobulin secretion by differentiated B-lymphocytes.

In contrast to pokeweed mitogen (PWM), S. aureus Cowan I (SAC) does not activate suppressor T-cells in cultures of human peripheral blood mononuclear cells, although the SAC induced response leading to the appearance of immunoglobulin secreting cells (ISC) is suppressed by PWM activated suppressor T-cells. Therefore, cultures co-stimulated by SAC and PWM were chosen to find out which stage of the SAC triggered B-cell response is controlled by PWM activated suppressor T-cells. By incorporation of [3H]thymidine and determination of B-cell number in culture it was shown that neither PWM itself nor PWM induced suppressor T-cells interfere with SAC induced B-cell proliferation. The final stages of B-cell maturation were monitored by parallel evaluation of cells producing immunoglobulins (cells with intracytoplasmic immunoglobulins) and secreting them (plaque assay). It was shown that PWM activated suppressor T-cells reduce the number of ISC more effectively than the number of immunoglobulin producing cells, indicating that these two features of differentiated B-lymphocytes may be independently controlled.

Elimination of monocytes from cultures activated with recall antigens.

Recent data provide evidence that antigen-specific CD4+ T-cell clones or antigen-activated T-cell lines can kill antigen-presenting cells (APC). We focused our studies on monocytes acting as APC in cultures of T cells freshly isolated from peripheral blood. The presence of monocytes in culture was monitored by their ability to emit light during phagocytosis of latex particles (latex-induced chemiluminescence). Using this approach as well as flow cytometry, evidence is presented that monocytes are eliminated from cultures with T cells activated with recall antigens (PPD or TT). The mechanism of monocyte elimination involved apoptosis as judged from in situ detection of DNA strand breaks by the terminal deoxynucleotidyl transferase assay. The antigen- but not lectin-dependent monocyte elimination was MHC-restricted and mediated by CD4+ T lymphocytes. This finding supports the hypothesis that elimination of APC is a general phenomenon during T-cell activation and may represent an important immunoregulatory mechanism.

Modulation of monocyte antigen-presenting capacity by tumour necrosis factor-alpha (TNF): opposing effects of exogenous TNF before and after an antigen pulse and the role of TNF gene activation in monocytes.

We have previously shown that exogenous human recombinant tumour necrosis factor-alpha (rTNF), added before an antigen pulse, enhanced antigen presentation by human blood monocytes. The present study shows that, surprisingly, rTNF added after an antigen (PPD) pulse inhibited, while anti-TNF monoclonal antibody (mAb) enhanced, antigen presentation. mAbs htr-9 against p55 TNF receptor type I (TNF-RI) abrogated rTNF enhancing effect on PPD presentation and decreased presenting activity of untreated monocytes while utr-1 mAb, against p75 TNF receptor type II (TNF-RII), reversed the inhibitory effect of rTNF given after antigen pulse. PPD and rTNF when added singly induced TNF-mRNA accumulation in monocytes. Pretreatment of monocytes with rTNF followed by a PPD pulse caused an enhancement of TNF-mRNA accumulation. However, when post-treatment with rTNF was applied to PPD-pulsed monocytes, then inhibition of TNF gene expression was seen. This may point to the role of endogenously generated TNF in regulation of antigen-presenting capacity of monocytes. These studies indicate that TNF is an important regulator of monocyte antigen-presenting capacity and that the level of TNF gene activation in monocytes may be associated with their ability to present nominal antigen.

Human MO subsets as defined by expression of CD64 and CD16 differ in phagocytic activity and generation of oxygen intermediates.

Phagocytosis and killing of microorganisms by reactive oxygen radicals are important defence mechanisms of the immune system and it was shown that human monocytes (MO) are heterogeneous in exerting these functions. Previously, we described that human peripheral blood MO consist of a major subset of Fc gamma-receptor-I (CD64)-positive cells exhibiting low accessory cell capacity but high phagocytic activity, and a minor subset of CD64-negative cells with dendritic cell (DC)-like high T cell accessory cell capacity but low phagocytic capacity. Recently, we could show that each subset itself further differs in the expression of the Fc gamma-receptor-III (CD16) and T cell accessory activities resulting in four different subsets: two CD16+ subsets (CD64+ or CD64-) with high T cell stimulation capacity and two CD16- subsets (CD64+ or CD64-) with low accessory activities. In the present study we demonstrate that these subsets also differ in their ability to phagocytose opsonized bacteria (S. aureus and E. coli) and in the generation of reactive oxygen species. Both CD64+ subsets (CD16+ or CD16-) exhibit high phagocytic activity accompanied by intracellular superoxide induction. Luminol-dependent (mainly myeloperoxidase (MPO)-mediated) chemiluminescence (CL) response to latex and FMLP (formylmethionylleucylphenylalanine) was also high in these cell populations. Phagocytic activity and modest CL response was shown in CD64-/CD16+ but not in CD64-/CD16- cells, indicating that each subset except for CD64-/CD16- cells may engulf bacteria and exhibit MPO activity. Taken together, these data demonstrate further heterogeneity of peripheral blood MO in both, phagocytic activity and generation of reactive oxygen species indicating differences between the four subsets in this kind of defence mechanisms against pathogens.

The role of monocytes in the induction and regulation of IFN-gamma production by lectin-activated human T lymphocytes.

The data presented show that the production of interferon gamma (IFN-gamma) by pokeweed mitogen (PWM)-activated T lymphocytes requires monocytes and that the amount of lymphokine produced depends on the number of monocytes present in the culture. Accessory function of monocytes was independent from their ability to secrete IL-1 but required cell-cell contact, since blocking of adhesion molecules reduced the IFN-gamma production. Furthermore, production of IFN by lectin-preactivated T lymphocytes could not be triggered by IL-2 but also required monocyte-T cell interaction.

Apoptosis in monocytes.

Apoptosis is a major mechanism for reducing acute inflammation by elimination of unwanted cellular responses causing tissue damage and monocytes (Mo) and macrophages (Mo) play an important role in these processes. Therefore, in the following we summarize how these cells may contribute to regulation of apoptosis of other cells and discuss the factors involved.

Alveolar macrophages of children suffering from recurrent infections of respiratory tract are less efficient in eliminating apoptotic neutrophils.

During bacterial infections of the respiratory tract, neutrophils (PMN) are recruited to the lung by various mechanisms, including production of interleukin-8 (IL-8) by alveolar macrophages (AM). After fulfilling their defense function, PMN become apoptotic and have to be disposed of by AM to prevent local damage to the lung tissue by oxygen species and proteolytic enzymes. We measured the levels of IL-8 in the bronchoalveolar lavage (BAL) and the ability of AM to engulf senescent PMN in a groups of children with and without recurrent infections of the respiratory tract. The IL-8 level was measured by enzyme-linked immunosorbent assay (ELISA). The phagocytosis of apoptotic neutrophils was evaluated microscopically by the presence of myeloperoxidase positive material in AM before and after 1 h of incubation with senescent PMN. The data show that children suffering from recurrent infections have increased IL-8 in BAL and that their AM have a lower ability to engulf apoptotic PMN in vitro. Furthermore, the proportion of annexin V-binding cells was higher in BAL of children with recurrent infections of the respiratory tract than in normal controls.

Detection of cancer cells in the blood by FACS sorting of CD45- cells.

We describe a simple and sensitive method for detection of low number of cancer cells in the blood. The method is based on FACS sorting of leukocytes labelled with anti-CD45 monoclonal antibody and examining CD45- cells by conventional cytology and immunostaining for cytokeratin 18. In a model study, cancer cells seeded at the frequency of 1 per 106 and 1 per 107 leukocytes were detected in CD45- population. Sensitivity of this method was comparable to reverse transcription polymerase chain reaction (RT-PCR) used for detection of cancer cells expressing CD44 variants-mRNA. In a pilot study, cancer cells were also isolated from the blood of some patients with locally advanced gastric cancer. This method may be useful for detection of circulating tumour cells in cancer patients.

Regulatory function of normal monocytes in the in vitro immunoglobulin production.

Induction of the immunoglobulin production by pokeweed mitogen (PWM) and S. aureus. Cowan I has been evaluated in cultures of human peripheral blood mononuclear cells gradually depleted of adherent cells. Partial depletion cells (monitoglobulins while almost complete depletion caused decreased production. These results suggest that an optimal balance of monocytes to lymphocytes is essential in the triggering of immunoglobulin production by human mononuclear cells (MNC) in vitro and that normal monocytes, when in excess, suppress in vitro differentiation of B lymphocytes into immunoglobulin secreting cells.

Attempts to evaluate the role of the third complement component (C3) and complement receptor lymphocytes (CRL"+") in the induction of the humoral immune response.

It has been shown that spleen and bone marrow complement receptor lymphocytes (CRL"+") are responsible for T-dependent anti-SRBC response. This has been shown by comparison of the T-dependent anti-SRBC and T-independent anti-SIII response in irradiated mice repopulated with normal or CRL depleted spleen cell populations. Unseparated spleen cells obtained from animals immunized 48 h before with SRBC, and CRL depleted cell population from animals receiving T-independent antigens (levan, SIII) were shown to be predominantly stimulated when uptake of isotope labelled leucine or tymidine was studied. It was also shown that triggering of enhanced antibody production by T cell derived signal generated during GvH reaction is C3 dependent.

The T-dependent and T-independent humoral immune response in cobra venom treated mice.

The humoral immune response to T-dependent antigens (SRBC, DNP5BSA, DNP50BSA, DNP-KLH) and T-independent antigens (SIII, Levan, DNP-SIII, DNP-Levan) was studied by hemolytic plaque assay in mice depleted of C3 by Cobra venom treatment. Suppression of the immune response in C3 depleted animals was not restricted only to T-dependent response and also the response to some T-dependent antigens (DNP50-BSA, DNP-KLH) was not suppressed under conditions of the experiment. Results are discussed in relation to postulated exclusive role of C3 in the induction of T-dependent immune response.

The reduced expression of HLA-class II antigens and adhesion molecules on monocyte surface after phagocytosis of bacteria.

The reduced expression of HLA-class II antigens (DQ and DP) and some intercellular adhesion molecules (CD11b, CD54 and CD58) was found on monocytes after phagocytosis of bacteria (S. aureus, E. coli, P. aeruginosa, S. enteritidis) but not of latex particles. In contrast, the expression of HLA-DR, CD11a and CD18 was not changed in the course of phagocytosis. The observed changes were related to the amount of phagocytosed bacteria but not to their viability or phagocytosis induced physical changes expressed as the reduction of FSC signal during flow cytometry analysis.

Influence of cobra venom (CoF) and pneumococcal S III capsular polysaccharide on immunologic humoral response and visceral distribution of antigens.

The immunologic response to sheep erythrocytes was compared in mice treated with Cobra venom (CoF) and with pneumococcal capsular antigen S III. Suppression of the immunologic response in both cases was attributed to the ability of both tested substances to activate the third component of complement. CoF and S III both changed the organ localization of labeled antigens capable of activating the complement system. No evidence for an alternative pathway by which S III could activate the complement system was found. On the basis of the literature data and experimental findings, the properties of CoF and S III were compared in the light of their ability to suppress the immunologic response.