Opsonization by anti-dsDNA antibodies of apoptotic cells in systemic lupus erythematosus

In order to asses the role of the soluble mediators of serum from patients with SLE in the apoptotic cell clearance, we measured the in vitro phagocytosis of apoptotic Jurkat cells by normal healthy donor macrophages in the presence of SLE patients' sera. A significant increase of the phagocytic index (NHD = 1.0 +/- 0.3; SLE = 1.9 +/- 0.6; p < 0.01) was to be observed in the presence of serum from patients with SLE. The increased phagocytic index correlated to the anti-dsDNA antibodies titers. We conclude that anti-dsDNA antibodies present in sera of patients with SLE favor the apoptotic cell phagocytosis by opsonization of the target cells. This may represent a deviation of the clearance process towards inflammation and a new pathologic feature of these autoantibodies in SLE.     

Differential infection of mononuclear phagocytes by Francisella tularensis: role of the macrophage mannose receptor

Francisella tularensis (Ft) is a Gram-negative bacterium and the causative agent of tularemia. It is well established that this organism replicates inside macrophages, but we are only beginning to understand this interface at the molecular level. Herein, we compared directly the ability of Ft subspecies holarctica live-vaccine strain to infect freshly isolated human peripheral blood monocytes, monocyte-derived macrophages (MDM), and cells of the murine macrophage cell line J774A.1 (J774). We now show that unopsonized bacteria infected human MDM fivefold more efficiently than monocytes or J774 cells in standard media. Moreover, enhanced infection of MDM was mediated, in part, by the macrophage mannose receptor (MR). Forming Ft phagosomes accumulated MR, and infection was inhibited by MR-blocking antibody or soluble mannan but not by the dectin-1 ligand laminarin. Up-regulation of MR in MDM (by exposure to interleukin-4) increased Ft phagocytosis, as did expression of MR in J774 cells. Conversely, opsonized Ft were ingested readily by monocytes and MDM. Medium supplementation with 2.5% fresh autologous serum was sufficient to confer opsonophagocytosis and CD11b accumulated in the membrane at sites of Ft engulfment. Infection of monocytes by opsonized Ft was nearly ablated by complement receptor 3 (CR3) blockade. Conversely, MDM used MR and CD11b/CD18 to ingest opsonized organisms. Altogether, our data demonstrate differential infection of mononuclear phagocytes by Ft and define distinct roles for MR and CR3 in phagocytosis.     

Phagocytosis of heat‐killed Staphylococcus aureus by eosinophils: comparison with neutrophils

Eosinophils are characterized by several functional properties, such as chemotaxis, adhesion, superoxide anion production, and degranulation. In this article, we have studied the role of bacterial ingestion by eosinophils in comparison with that by neutrophils. Eosinophils and neutrophils were purified by using the Percoll gradient method followed by selection with CD16‐coated immunomagnetic beads and centrifugation through a Ficoll‐Hypaque gradient combined with dextran sedimentation, respectively. Both cells were preincubated with anti‐FcγRIIa mAb (CD32 mAb), anti‐FcγRIIIb mAb (CD16 mAb), anti‐CR3 (CD11b mAb), or anti‐CR1 (CD35 mAb) before being examined for phagocytosis of opsonized heat‐killed Staphylococcus aureus (S. aureus). Phagocytosis and production of hydrogen peroxide were simultaneously measured by flow cytometry using S. aureus labeled with propidium iodide and stained with 2′,7′‐dichlorofluorescein diacetate. Eosinophils showed significantly lower activity than neutrophils in both phagocytosis and hydrogen peroxide production. Phagocytosis by both cells was decreased by heat‐inactivated serum. Phagocytosis by neutrophils was significantly inhibited by CD16 mAb and CD32 mAb, whereas that by eosinophils was only inhibited by CD35 mAb. Whereas the mechanism of phagocytosis by neutrophils was mediated by CD16 and CD32, that of eosinophils was modulated by complement receptor 1 (CD35).     

Anti-CD45 isoform antibodies enhance phagocytosis and gene expression of IL-8 and TNF-alpha in human neutrophils by differential suppression on protein tyrosine phosphorylation and p56lck tyrosine kinase

To determine the biological functions of membrane expressed CD45 isoforms on polymorphonuclear neutrophils (PMN), the monoclonal IgG F(ab')2 antibody against CD45, CD45RA or CD45RO was used as surrogate ligand for binding with these molecules on PMN. We found 99.5 +/- 3.2%, 42.3 +/- 5.8% and 96.7 +/- 2.6% PMN expressed CD45, CD45RA and CD45RO molecules on the cell surface, respectively. The interaction of CD45, CD45RA or CD45RO with its specific antibody on PMN enhanced phagocytosis markedly (34-83% increase), mainly via increased expression of complement receptor type 3 (CR3, CD11b) on the cells. The production of IL-8 by PMN was also increased significantly after binding with antibodies (anti-CD45 > anti-CD45RO > anti-CD45RA). Anti-CD45RA and anti-CD45RO, but not anti-CD45, enhanced TNF-alpha mRNA expression and decreased protein tyrosine phosphorylation of PMN. However, only anti-CD45RO suppressed Src family protein tyrosine kinase p56lck expression in the cells. These results suggest that the cross-linking of CD45 isoforms by their specific antibodies stimulated different PMN activities by differential suppression on protein tyrosine phosphorylation and Src family tyrosine kinase p56lck.     

Immunohistochemical changes in sigmoid colon after allogeneic and autologous bone marrow transplantation.

AIM--To determine whether there are characteristic immunohistological changes in the colonic mucosa in acute graft versus host disease (GvHD). METHODS--Consecutive allogeneic (n = 11) and autologous (n = 11) bone marrow transplant recipients underwent endoscopic biopsy of sigmoid mucosa before transplant and on day 30 post-transplant. Immunohistochemical staining and quantitation of intraepithelial and lamina propria mononuclear cells were undertaken using a panel of monoclonal antibodies and a Streptavidin-biotin alkaline phosphatase staining technique. RESULTS--In the allogeneic group (nine of whom had clinical acute GvHD) there was a fivefold increase in lamina propria CD16+ mononuclear cells (3.1 +/- 4.3 to 17.0 +/- 12.2 per 100 lamina propria nucleated cells), compared with autologous transplant recipients in whom this rise was twofold (5.5 +/- 4.6 to 10.6 +/- 7.1 per 100 lamina propria nucleated cells). The CD16+ mononuclear cells had morphological appearances of tissue macrophages, but in neither the allogeneic nor autologous groups was there an increase in total macrophage numbers (CD14+). In patients with acute GvHD the lamina propria CD4+:CD8+ lymphocyte ratio fell (1.97 +/- 1.12 to 1.07 +/- 1.01), primarily because of a fall in the number of lamina propria CD4+ lymphocytes. In both allogeneic and autologous groups there was a fall in intraepithelial lymphocyte numbers, but there was no change in CD19+ (B cell), CD25+ (interleukin-2 receptor positive) or CD56+ (natural killer) cell numbers. CONCLUSION--Following bone marrow transplantation, there appears to be upregulation of lamina propria tissue macrophage CD16 (an Fc receptor for IgG), a change which is more noticeable after allogeneic transplantation and which may be related to the development of acute GvHD. In patients with acute GvHD there was a fall in the lamina propria CD4+:CD8+ lymphocyte ratio. If these changes are functionally important, they may have significant implications for understanding the pathogenesis of GvHD.     

Role and modulation of CD16 expression on eosinophils by cytokines and immune complexes

BACKGROUND: Blood eosinophils express CD16 on their surface when stimulated in vitro with platelet-activating factor or IFNgamma. Transient expression of CD16 is also observed in vivo following aeroallergen challenge of asthmatic subjects. The present work is aimed at evaluating the possible mechanisms modulating eosinophil expression of CD16 and the biological functions of this receptor. METHODS: First, purified blood eosinophils were incubated with IL-1beta, IL-2, IL-4, IL-5, IL-9 or IL-16, GM-CSF, IFNgamma, eotaxin or 5-oxo-ETE and CD16 expression was measured. Second, the capacity of CD16 to mediate degranulation induced by IgG immune complexes (IC) was evaluated in eosinophils with low and high CD16 expression. Finally, serum allergen-specific IgE and IgG, and total IgE levels were measured at baseline in allergic asthmatics and correlated with changes observed in blood eosinophil CD16 expression (DeltaCD16) following allergen challenge. RESULTS: Only IFNgamma and IL-2 significantly increased the number of CD16+ eosinophils, respectively, 37 +/- 10% (p = 0.0038) and 38 +/- 8% (p = 0.0006), compared to control, 7 +/- 2%. IgG IC induced degranulation in eosinophils with low and high CD16 expression and monoclonal anti-CD16 and anti-CD32 antibodies inhibited this. IgG IC increased eosinophil CD16 expression (14 +/- 6%, p = 0.0008) and this effect was blocked by pretreatment with anti-CD32 antibodies. DeltaCD16 following allergen challenge correlated with the specific IgG/total IgE ratio (r(2) = 0.41, p = 0.036). CONCLUSION: These data suggest that formation of IgG IC is associated with surface eosinophil CD16 expression in asthma and that CD16 in cooperation with CD32 mediates IC-induced degranulation.     

Serum-derived protein S binds to phosphatidylserine and stimulates the phagocytosis of apoptotic cells

Rapid phagocytosis of apoptotic cells is thought to limit the development of inflammation and autoimmune disease. Serum enhances macrophage phagocytosis of apoptotic cells. Here we identified protein S as the factor responsible for serum-stimulated phagocytosis of apoptotic cells. Protein S is best known for its anti-thrombotic activity, serving as a cofactor for protein C. Purified protein S was equivalent to serum in its ability to stimulate macrophage phagocytosis of apoptotic lymphoma cells, and immunodepletion of protein S eliminated the prophagocytic activity of serum. Protein S acted by binding to phosphatidylserine expressed on the apoptotic cell surface. Protein S is thus a multifunctional protein that can facilitate clearance of early apoptotic cells in addition to regulating blood coagulation.     

The role of Fcgamma receptor polymorphisms and C3 in the immune defence against Neisseria meningitidis in complement-deficient individuals

Individuals with either a late (C5-9) complement component deficiency (LCCD) or properdin deficiency are at increased risk to develop meningococcal disease, often due to serogroups W135 and Y. Anti-meningococcal defence in both LCCD persons and properdin-deficient individuals without bactericidal antibodies depends mainly on phagocytosis. Three types of opsonin receptors are involved in phagocytosis by polymorphonuclear cells (PMN). These represent the polymorphic FcgammaRIIa (CD32) and FcgammaRIIIb (CD16b) receptors, and the C3 receptor CR3 (CD11b/CD18). When the distribution of FcgammaRIIa and FcgammaRIIIb allotypes was assessed in 15 LCCD and in 15 properdin-deficient patients with/without previous meningococcal disease, we found the combination of FcgammaRIIa-R/R131 with FcgammaRIIIb-NA2/NA2 allotypes to be associated with previous meningococcal disease (odds ratio 13.9, Fisher's test P = 0.036). No such relation was observed in the properdin-deficient patients. The importance of FcgammaRIIa allotypes was also demonstrated using in vitro phagocytosis assays. PMN from FcgammaRIIa-R/R131 homozygous donors internalized IgG2 opsonized meningococci W135 significantly (P < 0.05) less than PMN from FcgammaRIIa-H/H131 donors. When properdin-deficient serum was tested, it was observed that reconstitution with properdin resulted in enhanced PMN phagocytosis of the W135 meningococci (P = 0.001). This enhanced phagocytosis was parallelled by an increase in C3 deposition onto the opsonized meningococci W135 (r = 0.6568, P = 0. 01). We conclude that the occurrence of meningococcal disease in LCCD patients is associated with certain FcgammaR allotypes. Properdin-deficient individuals are susceptible to meningococcal disease because of an insufficient C3 deposition on the surface of meningococci, resulting in insufficient phagocytosis.     

High serum levels of CD8 antigen in primary biliary cirrhosis: a possible cause of suppressor cell dysfunction?

Reduced suppressor cell number and function have been described in a number of autoimmune diseases and this may contribute to pathogenesis. Suppressor cell function depends upon the interaction of the CD8 antigen expressed on suppressor cells with other limbs of the immune system. Recently, soluble membrane antigens including CD8 have been identified in serum and it is possible that the loss of such antigens from viable cells could result in functional deficit. In order to examine whether the decreased suppressor cell function reported in autoimmune type of chronic liver disease is associated with soluble serum CD8 levels, sera from 23 patients with primary biliary cirrhosis (PBC), 12 with autoimmune chronic active hepatitis (AI-CAH) and 21 healthy controls were tested using a commercially available enzyme immunoassay. The proportion of cells expressing the CD8 antigen and the intensity of its display were also determined using an immunofluorescent technique and an ELISA, respectively, for 12 PBC and 10 healthy controls. The soluble serum CD8 levels were significantly higher in PBC (mean U/ml +/- s.d., 777 +/- 331), and AI-CAH (575 +/- 291) than controls (322 +/- 115) (P less than 0.001 and P = 0.004, respectively). While the intensity of CD8 antigen expression on suppressor/cytotoxic populations was not significantly different in PBC (347 +/- 125 per 10(4) cells) compared with controls (441 +/- 206), the mean proportion of CD8 positive cells was significantly less in PBC (14.1 +/- 6.8%) than controls (20 +/- 4.7%) (P less than 0.05). These data suggest that the apparent reduction in suppressor cell number found for patients with PBC and AI-CAH may be a consequence of the shedding or secretion of CD8 antigen from cell membrane of CD8 positive lymphocyte. It is also possible that the loss of this antigen is responsible for the reduced suppressor cell function seen in these conditions.     

The role of complement receptor type 1 (CR1, CD35) in determining the cellular distribution of opsonized immune complexes between whole blood cells: kinetic analysis of the buffering capacity of erythrocytes.

Erythrocytes (E) express complement receptor, type 1 (CR1, CD35), by which they bind opsonized immune complexes (IC) in competition with leucocytes expressing higher numbers of CR1 as well as other complement- and Fc-receptors. This may prevent inappropriate activation of phagocytic cells. We examined the distribution on whole blood cells of preformed tetanus toxoid (TT)/human anti-TT IC, opsonized in situ in 80% autologous serum. Binding to E occurred rapidly and reflected the kinetics of C3-fragment incorporation into the IC. Among eight donors, expressing 180-361 CR1 per E. > 90% of the cell-bound IC were associated with E from 1 to 5 min of incubation, decreasing to 12 +/- 13% after 40 min. Upon comparison of the IC-binding to leucocytes in whole blood with that of isolated leucocytes we found that E, despite their extensive early complex uptake, only reduced the IC-deposition on polymorphonuclear leucocytes (PMN) by 61 +/- 26% after 30 seconds of incubation and 47 +/- 14% after 5 min. During the subsequent 10 min, this buffering capacity of E was essentially abolished E restricted the initial IC-binding to B cells by 73 +/- 19%, but from 3 min of incubation the presence of E promoted, in a CR1-dependent manner, a progressive uptake via CR2 by the B cells. CR1 was the dominant receptor in the early IC-uptake by B cells as well as PMN and monocytes, since CR1-blockade inhibited the initial IC-uptake by these populations in a preparation of isolated leucocytes suspended in serum by > or = 84% after 30 seconds of incubation. We conclude, that E exert a substantial buffering effect on the IC-deposition on PMN, monocytes and B cells, while CR1 is the dominant receptor in the uptake by these cells. However, this effect is short-lived and less than expected from the proportion of IC bound to E. Moreover, E are efficient processors of IC-attached C3b/iC3b fragments to C3dg as indicated by a pronounced enhancement by E of IC-uptake via CR2 on B cells. We propose that this mechanism may play a role in preventing phagocyte activation via CR3.     

The selective engulfment of apoptotic bodies by dendritic cells is mediated by the αvβ3 integrin and requires intracellular and extracellular calcium

Dendritic cells derived in vitro from monocytes are known to be poor phagocytes. Here we show that, unlike macrophages, monocyte-derived dendritic cells indeed fail to take up opsonized particles or necrotic cells; however, apoptotic bodies are efficiently engulfed by dendritic cells. The temperature dependence and the sensitivity to cytochalasin D indicate that the apoptotic body engulfment is representative of early stages of phagocytosis. Inhibition studies with ligands for surface molecules involved in recognition of apoptotic bodies, such as vitronectin receptor, CD36 and phosphatidylserine receptor, revealed that apoptotic body engulfment by dendritic cells is mediated preferentially by the vitronectin receptor αvβ3, while all the receptors, with different efficiency, are engaged in phagocytosis of apoptotic bodies by macrophages. The interaction between apoptotic bodies and dendritic cells elicits a rise in intracellular free calcium concentration ([Ca²⁺]_i) which is essential for the process of engulfment. Either intra- or extracellular Ca²⁺ buffering inhibits apoptotic body engulfment by dendritic cells and [Ca²⁺]_i increases, indicating the involvement of both intra-and extracellular Ca²⁺. In contrast, Ca²⁺ mobilization is dispensable for macrophage phagocytosis of apoptotic bodies. The different requirements of Ca²⁺ in macrophages and dendritic cells is possibly due to the differential usage of phagocytic receptors (CD36 vs. αvβ3) and might reflect different fates of apoptotic bodies in the two cell types.     

Liver-infiltrating T helper cells in autoimmune chronic active hepatitis stimulate the production of autoantibodies against the human asialoglycoprotein receptor in vitro.

Autoantibodies against the human asialoglycoprotein receptor (ASGPR) occur in the sera of patients with autoimmune liver disorders. Liver-infiltrating T cell clones that specifically recognize the ASGPR have been described in patients with autoimmune chronic active hepatitis (AI-CAH) and primary biliary cirrhosis (PBC). Recently, we have shown that peripheral blood mononuclear cells (PBMC) from patients with AI-CAH or PBC but not chronic viral hepatitis secreted anti-ASGPR antibodies in vitro. In this study we characterized the influence of liver-infiltrating T cells on the secretion of ASGPR-specific autoantibodies by autologous B cells in cell culture supernatants. T cell clones from liver biopsies of three patients with chronic autoimmune liver disorders (one with AI-CAH, two with PBC) were isolated and investigated for their proliferative response to soluble ASGPR and their helper function provided to autoantibody-secreting B lymphocytes. PBMC from these patients secreted autoantibodies spontaneously in their cell culture supernatants and showed a proliferative response to ASGPR. T cell-depleted PBMC, however, lacked spontaneous antibody secretion. Four CD4+CD8- liver-infiltrating T cell clones showed a proliferative response to ASGPR and also induced spontaneous anti-ASGPR antibody production in cell culture supernatants when added to autologous T cell depleted PBMC. Activated supernatants of these T cell clones failed to induce antibody production. None of seven CD4+CD8- and two CD4-CD8+ T cell clones non-responding to ASGPR provided this help for antibody secretion. Anti-ASGPR secretion in vitro could not be inhibited by the addition of MoAbs raised against monomorphic determinants on HLA class II molecules. The addition of purified ASGPR or polyclonal-activating pokeweed mitogen showed no influence on the production of autoantibodies in these cultures. These data show that B lymphocytes require T cell help for the production of ASGPR-specific antibodies. This help can be provided by ASGPR-responsive T helper cells via cellular interactions.     

Evidence for the Role of CR1 (CD35), in addition to CR2 (CD21), in Facilitating Infection of Human T Cells with Opsonized HIV

Complement activation by HIV results in the binding of C3 fragments to the gp160 complex and enhanced infection of C3 receptor-bearing target cells. We have studied complement-mediated enhancement of infection of the human CD4-positive T-cell line HPB-ALL which expresses the CR1 (CD35) and CR2 (CD21) receptors for C3. CR1 and CR2 are present on 15% and 40% of normal peripheral blood CD4-positive T lymphocytes respectively. Opsonization of the virus with complement resulted in a 3- to 10-fold enhancement of infection of HPB-ALL cells, as assessed by measuring the release of p24 antigen in culture supernatants throughout the culture period. Blockade of CR2 with cross-linked anti-CR2 monoclonal antibodies decreased infection to the level observed with unopsonized virus. Blocking CR1 reduced complement-mediated infection by 50–80%. Experiments using serum deficient in complement factor I demonstrated that CR1 mediates the interaction between opsonized virus and T cells in addition to its ability to serve as a cofactor for the cleavage of C3b into smaller fragments that interact with CR2. A requirement for CD4 in complement-mediated enhancement of infection was observed with HIV-1 Bru but not with HIV-1 RF. Thus, CR1 and CR2 contribute in an independent and complementary fashion to penetration of opsonized virus into complement receptor-expressing T cells. Involvement of CD4 in infection with opsonized virus depends on the viral strain.     

Opsonization of Staphylococcus aureus protects endothelial cells from damage by phagocytosing polymorphonuclear leukocytes.

When phagocytosis of Staphylococcus aureus by human polymorphonuclear leukocytes (PMN) takes place on the surface of cultured human endothelial cells, the endothelial monolayers are damaged by lysosomal enzymes that are released by the PMN. Because PMN can phagocytose opsonized as well as unopsonized staphylococci on an endothelial surface, we studied the role of bacterial opsonization in the damage caused to the endothelium. Phagocytosis of unopsonized S. aureus was accompanied by greater damage (expressed as the percentage of the endothelial cells detached from the culture plates) of the monolayers than was phagocytosis of opsonized S. aureus: 52 +/- 10% and 24 +/- 7%, respectively, after 30 min of phagocytosis and 73 +/- 5% and 50 +/- 6%, respectively, after 60 min of phagocytosis. When correlated to the amount of phagocytosis, this difference was even greater (uptake was 35 +/- 4% for unopsonized S. aureus and 56 +/- 5% for opsonized S. aureus after 30 min and 42 +/- 3% and 60 +/- 5%, respectively, after 60 min). Total release of lysozyme and myeloperoxidase and generation of superoxide anion were the same during phagocytosis of opsonized or unopsonized staphylococci. Adherence of PMN to the endothelial cells was greater during phagocytosis of unopsonized S. aureus: 42 +/- 4% verus 27 +/- 3% during phagocytosis of opsonized staphylococci. Possibly, increased adherence of the PMN resulted in a locally higher concentration of enzymes which induced more damage. We conclude that opsonization of bacteria not only improves bacterial uptake, but also protects bystander cells from damage by the phagocytosing PMN.     

An assay for the quantitative measurement of in vitro phagocytosis of early apoptotic thymocytes by murine resident peritoneal macrophages

Research into the mechanisms by which apoptotic cells are phagocytosed has grown considerably over recent years, together with a growing appreciation of the importance of clearance of redundant cells for tissue homeostasis. However, studies addressing the efficacy of phagocytosis have been rare. The few studies reported to date were either attempts to determine apoptotic cell clearance from the circulation or were focused on clearance in inflammation. We now describe an in vitro assay which permits the quantitative measurement of phagocytosis of apoptotic cells by murine resident peritoneal macrophages. The apoptotic cells used in the assay were murine thymocytes incubated with dexamethasone for only 3 h. Most apoptotic thymocytes were annexin V positive and propidium iodide negative and therefore still in the earlier stages of apoptosis. The assay was completed 7 h after the isolation of both macrophages and thymocytes, while macrophage culture time was only 4 h. Because of this short-term culture it is likely that the resident peritoneal macrophages largely maintained their in vivo phenotype. Using BALB/c macrophages and thymocytes, the maximal in vitro phagocytosis exceeded five thymocytes per macrophage in 1 h and two of these thymocytes were taken up within 10 min. Therefore, in vitro phagocytosis by resident peritoneal macrophages was rapid and of high capacity, as it is postulated to be in vivo. Under selected conditions, the mean uptake was 4.45+/-0.70 (mean +/- SD, n = 31) thymocytes per macrophage in 1 h. The inter-assay coefficient of variation, also representing the biological variability, was found to be 15.7%. The average intra-assay coefficient of variation was 13.6%. This assay permits comparisons of phagocytic efficacy between different strains of mice in vitro. In addition, a method of preparation is described which allows long-term storage of experimental results. Finally, our data suggests that internalization, but not binding of apoptotic cells to short-term cultured resident peritoneal macrophages, is critically dependent on the presence of serum. This allows separate analysis of binding and internalization of apoptotic cells with the assay, without the necessity to use agents blocking internalization.     

Apoptotic killing and phagocytosis of host cells by the parasite Entamoeba histolytica

The ability of Entamoeba histolytica to kill and phagocytose host cells correlates with parasite virulence. This study addressed the role of apoptotic cell killing and host cell phosphatidylserine exposure in the subsequent phagocytosis of Jurkat T cells by E. histolytica. Ingested host cells were apoptotic, as evidenced by the activation of caspase 3 in 88% +/- 3% (mean and standard deviation [SD] of the mean) of Jurkat cells engulfed by E. histolytica; ingested cells without detectable active caspase 3 were already disrupted and partially digested. That apoptotic cell killing preceded phagocytosis was supported by the demonstration that a higher percentage of amebae ingested apoptotic cells than ingested healthy cells (62% +/- 7% versus 30% +/- 9%, respectively [mean and SD]) (P = 0.008). E. histolytica also ingested apoptotic Jurkat cells more rapidly than necrotic control cells (8.5% +/- 0.4% versus 3.5% +/- 0.7%, respectively [mean and SD]) (P < 0.001). The inhibition of amebic cytotoxicity with D-galactose (which blocks the amebic Gal/GalNAc lectin) blocked the phagocytosis of healthy cells by greater than 80%, providing further evidence that apoptosis preceded engulfment. In contrast, D-galactose blocked the phagocytosis of already apoptotic cells by only 40%, implicating an additional host ligand (besides D-galactose) in amebic engulfment of apoptotic cells. The most characteristic surface change on apoptotic cells is phosphatidylserine exposure. Consistent with a role for host cell phosphatidylserine exposure in amebic ingestion of killed cells, Jurkat cell phosphatidylserine was exposed during incubation with E. histolytica (27% +/- 1% [mean and SD] specific increase at 30 min) (the P value versus the control was 0.0003). Approximately 50% more amebae ingested viable Jurkat cells expressing phosphatidylserine on the outer leaflet of the plasma membrane than ingested control cells (30.3% +/- 2.2% versus 19.8% +/- 1.9%, respectively [mean and SD]) (P = 0.003). By analogy with phagocytic clearance during apoptosis in metazoans, amebic apoptotic host cell killing followed by phagocytosis may limit inflammation and enable amebae to evade the host immune response.     

Apoptotic DNA binds to HLA class II molecules inhibiting antigen presentation and participating in the development of anti-inflammatory functional behavior of phagocytic macrophages

Resident macrophages are mainly responsible for the clearance of apoptotic cells from tissue by phagocytosis. Phagocytosis of apoptotic cells is not accompanied by activation of inflammatory mechanisms, unlike what happens when necrotic phenomena occur. We analyzed the effect of phagocytosis of apoptotic bodies on macrophage cell functions. After phagocytosis of apoptotic cells macrophages were unable to present an exogenous antigen to autologous antigen-specific T-cell lines. The inhibition was mediated by different mechanisms including binding of apoptotic DNA to human leukocyte antigen (HLA) class II molecules of macrophages, decreased expression of co-stimulatory molecules and increased secretion of tumor growth factor beta (TGFbeta). When dendritic cells were cultured with macrophages phagocytosing apoptotic cells, or with their supernatant, impaired dendritic cell antigen presenting activity and reduced tumor necrosis factor alpha (TNFalpha) secretion were found. Our results suggest that: (1) the phagocytosis of apoptotic bodies inhibits macrophage antigen presentation; (2) such inhibition is mediated by the binding of apoptotic DNA to macrophage HLA class II molecules as well as by the activation of biological mechanisms that induce an anti-inflammatory functional behavior in macrophages; and (3) macrophages phagocytosing apoptotic cells inhibit antigen presentation of neighboring dendritic cells via TGFbeta secretion. These events are likely related to the preservation of healthy tissues from the onset of inflammation.     

Anti-C1q autoantibodies from active lupus nephritis patients could inhibit the clearance of apoptotic cells and complement classical pathway activation mediated by C1q in vitro

Anti-C1q antibodies are prevalent in patients with active lupus nephritis and were found to be closely associated with renal involvement and predictive for a flare of nephritis. However, the pathogenesis of anti-C1q antibodies involved in human lupus nephritis remains unclear. C1q, which plays a key role in apoptotic cell and immune complex removal, is a very important functional molecule in the pathogenesis of SLE. The aim of this study was to investigate the influence of anti-C1q autoantibodies from active lupus nephritis patients on the bio-functions of C1q in vitro. We purified IgG autoantibodies against C1q from lupus nephritis patients, and found that they could recognize C1q bound on early apoptotic cells at 30 μg/ml, and could significantly decrease the phagocytosis by macrophages of early apoptotic cells opsonized by 50 μg/ml C1q in comparison with normal IgG. Levels of circulating immune complexes of the ten patients were measured by a circulating immune complexes (CIC)-C1q Enzyme Immunoassay Kit. Anti-C1q autoantibodies affinity purified by microtiter plates could significantly inhibit the deposition of C3c on CIC-C1q in a dose dependent manner in comparison with IgG from 10 healthy blood donors. The binding of opsonized immune complexes to RBCs was significantly inhibited by anti-C1q autoantibodies purified by microtiter plates in a dose dependent manner. Our observations suggest that serum anti-C1q autoantibodies from active lupus nephritis patients could interfere with some biological function of C1q in vitro.     

Autoimmune Thyroid Disease

In order to asses the role of the soluble mediators of serum from patients with SLE in the apoptotic cell clearance, we measured the in vitro phagocytosis of apoptotic Jurkat cells by normal healthy donor macrophages in the presence of SLE patients' sera. A significant increase of the phagocytic index (NHD = 1.0 ± 0.3; SLE = 1.9 ± 0.6; p < 0.01) was to be observed in the presence of serum from patients with SLE. The increased phagocytic index correlated to the anti-dsDNA antibodies titers. We conclude that anti-dsDNA antibodies present in sera of patients with SLE favor the apoptotic cell phagocytosis by opsonization of the target cells. This may represent a deviation of the clearance process towards inflammation and a new pathologic feature of these autoantibodies in SLE.     

Opsonin-independent phagocytosis of group B streptococci: role of complement receptor type three.

The role of complement receptor type 3 (CR3) in nonopsonic recognition of group B streptococci (GBS) by macrophages was investigated. Monoclonal anti-CR3 (anti-Mac-1) inhibited phagocytosis of GBS strains by as much as 50% in serum-free cultures of both mouse peritoneal macrophages and the macrophage cell line PU5-1.8. GBS uptake was unaffected by the presence of anti-C3 or salicylhydroxamate, an inhibitor of the covalent binding reaction of C3. Soluble antibodies to LFA-1 or to the common beta-chain (CD18) of the LFA-1/CR3/p150,95 family of cell adhesion molecules did not inhibit GBS uptake. Down-modulation of surface Mac-1 on macrophages following adherence to anti-Mac-1- or anti-CD18-coated surfaces also inhibited uptake of GBS. Further evidence for GBS interaction with CR3 was demonstrated by reduction of EC3bi rosette formation in macrophages adherent to GBS-coated plates. These studies suggest that GBS can interact with macrophage CR3, promoting phagocytosis in a C3-independent fashion. In the absence of specific immunity in neonates, this recognition mechanism may be a significant virulence determinant for GBS which poorly activate the alternate complement pathway.