Stylianos Bournazos

Apoptotic human cells inhibit migration of granulocytes via release of lactoferrin

Apoptosis is a noninflammatory, programmed form of cell death. One mechanism underlying the non-phlogistic nature of the apoptosis program is the swift phagocytosis of the dying cells. How apoptotic cells attract mononuclear phagocytes and not granulocytes, the professional phagocytes that accumulate at sites of inflammation, has not been determined. Here, we show that apoptotic human cell lines of diverse lineages synthesize and secrete lactoferrin, a pleiotropic glycoprotein with known antiinflammatory properties. We further demonstrated that lactoferrin selectively inhibited migration of granulocytes but not mononuclear phagocytes, both in vitro and in vivo. Finally, we were able to attribute this antiinflammatory function of lactoferrin to its effects on granulocyte signaling pathways that regulate cell adhesion and motility. Together, our results identify lactoferrin as an antiinflammatory component of the apoptosis milieu and define what we believe to be a novel antiinflammatory property of lactoferrin: the ability to function as a negative regulator of granulocyte migration.

Functional and clinical consequences of Fc receptor polymorphic and copy number variants

Receptors for immunoglobulins (Fc receptors) play a central role during an immune response, as they mediate the specific recognition of antigens of almost infinite diversity by leucocytes, thereby linking the humoral and cellular components of immunity. Indeed, engagement of Fc receptors by immunoglobulins initiates a range of immunoregulatory processes that might also play a role in disease pathogenesis. In the circulation, five main types of immunoglobulins (Ig) exist – namely IgG, IgA, IgE, IgM and IgD and receptors with the ability to recognize and bind to IgG (Fcγ receptor family), IgE (FcεRI and CD23), IgA (CD89; Fcα/µR) and IgM (Fcα/µR) have been identified and characterized. However, it is astonishing that nearly all the known human Fc receptors display extensive genetic variation with clear implications for their function, thus representing a substantial genetic risk factor for the pathogenesis of a range of chronic inflammatory disorders.

Glucocorticoids Induce Protein S-Dependent Phagocytosis of Apoptotic Neutrophils by Human Macrophages

During resolution of an inflammatory response, recruited neutrophil granulocytes undergo apoptosis and are removed by tissue phagocytes before induction of secondary necrosis without provoking proinflammatory cytokine production and release. Promotion of physiological neutrophil clearance mechanisms may represent a viable therapeutic strategy for the treatment of inflammatory or autoimmune diseases in which removal of apoptotic cells is impaired. The mechanism underlying enhancement of macrophage capacity for phagocytosis of apoptotic cells by the powerful anti-inflammatory drugs of the glucocorticoid family has remained elusive. In this study, we report that human monocyte-derived macrophages cultured in the presence of dexamethasone exhibit augmented capacity for phagocytosis of membrane-intact, early apoptotic cells only in the presence of a serum factor. Our results eliminate a role for a number of potential opsonins, including complement, pentraxin-3, and fibronectin. Using ion-exchange and gel filtration chromatography, we identified a high molecular mass serum fraction containing C4-binding protein and protein S responsible for the augmentation of phagocytosis of apoptotic neutrophils. Because the apoptotic neutrophils used in this study specifically bind protein S, we suggest that glucocorticoid treatment of macrophages induces a switch to a protein S-dependent apoptotic cell recognition mechanism. Consistent with this suggestion, pretreatment of macrophages with Abs to Mer tyrosine kinase, a member of the Tyro3/Axl/Mer family of receptor tyrosine kinases, prevented glucocorticoid augmentation of phagocytosis. Induction of a protein S/Mer tyrosine kinase-dependent apoptotic cell clearance pathway may contribute to the potent anti-inflammatory effects of glucocorticoids, representing a potential target for promoting resolution of inflammatory responses.

Monocyte Functional Responsiveness After PSGL-1–Mediated Platelet Adhesion Is Dependent on Platelet Activation Status

Objective—Acute coronary diseases are characterized by elevated levels of circulating platelet-leukocyte complexes, raising the possibility that proinflammatory processes might be initiated in leukocytes after platelet adhesion. Here we examined the mechanism of platelet binding to polymorphonuclear leukocytes, monocytes, and monocyte subsets and investigated the potential functional consequences of monocyte binding to minimally activated or thrombin-activated platelets. Methods and Results—In this article, we describe key differences in terms of stability of PSGL-1–mediated interaction of platelets with monocytes and polymorphonuclear leukocytes and a small but significant difference in platelet binding to monocyte subsets (CD14high and CD14low/HLA-DRhigh). We also report differential effects of platelet binding on monocyte functional responses between minimally and thrombin-activated platelets. In particular, monocyte CD11b expression and release of proinflammatory cytokines, like interleukin 1&bgr; and tumor necrosis factor &agr;, were significantly upregulated on adhesion of stimulated platelets, whereas unstimulated platelets had no effect. Moreover, binding of unstimulated, but not of thrombin-activated, platelets to monocytes had no impact on NF-&kgr;B activity, monocyte migration, and induction of apoptosis in the absence of survival factors. Conclusions—Our data suggest that in the absence of overt activation, PSGL-1–P-selectin–dependent platelet binding to monocytes represents a normal physiological process with little impact on the potential of monocytes to cause vascular injury.

Association of FcgammaRIIa (CD32a) with lipid rafts regulates ligand binding activity.

Binding of Igs to myeloid cells via FcR is a key event in the control of innate and acquired immunity. FcγRIIa (CD32a) is a receptor for multivalent IgG expressed predominantly by myeloid cells, and its association with microdomains rich in cholesterol and sphingolipids, termed as lipid rafts, has been reported to be essential for efficient signaling. However, for many myeloid cell types, ligand binding to CD32a is suppressed by as yet undefined mechanisms. In this study, we have examined the role of CD32a-lipid raft interactions in the regulation of IgG binding to CD32a. Disruption of lipid raft structure following depletion or sequestration of membrane cholesterol greatly inhibited CD32a-mediated IgG binding. Furthermore, specific CD32a mutants, which show reduced association with lipid rafts (A224S and C241A), displayed decreased levels of IgG binding compared with wild-type CD32a. In contrast, constitutively lipid raft-associated CD32a (GPI-anchored CD32a) exhibited increased capacity for IgG binding compared with the full-length transmembrane CD32a. Our findings clearly suggest a major role for lipid rafts in the regulation of IgG binding and, more specifically, that suppression of CD32a-mediated IgG binding in myeloid cells is achieved by receptor exclusion from lipid raft membrane microdomains.

Choice of Anticoagulant Critically Affects Measurement of Circulating Platelet-Leukocyte Complexes

In circulation, platelets adhere to leukocytes forming relatively stable complexes that have been reported to be elevated in cases of unstable angina, myocardial infarction, coronary artery disease, and postangioplasty restenosis.1–10 For this reason, measurement of circulating platelet-leukocyte complexes has been proposed as an early and accurate marker of in vivo platelet activation and myocardial injury after infarction.9,10 Increased levels of such complexes have also been noted in a range of chronic inflammatory diseases, including rheumatoid arthritis, end-stage renal failure, type I diabetes, and systemic lupus erythematosus.11–13 In the majority of published studies that have examined platelet-monocyte or platelet-polymorphonuclear (PMN) leukocyte complexes in human peripheral venous blood, sodium citrate (0.32 to 0.38%), a calcium-depleting agent, has been used as the blood anticoagulant. Because platelet adhesion to leukocytes is predominantly mediated by calcium-dependent interactions between platelet P-selectin and its leukocyte counter-receptor, P-selectin glycoprotein ligand-1 (PSGL-1),14 we aimed to determine whether calcium depletion by sodium citrate could affect …

Association of FcγRIIa R131H polymorphism with idiopathic pulmonary fibrosis severity and progression

BackgroundA significant genetic component has been described for idiopathic pulmonary fibrosis (IPF). The R131H (rs1801274) polymorphism of the IgG receptor FcγRIIa determines receptor affinity for IgG subclasses and is associated with several chronic inflammatory diseases. We investigated whether this polymorphism is associated with IPF susceptibility or progression.MethodsIn a case-control study, we compared the distribution of FcγRIIa R131H genotypes in 142 patients with IPF and in 218 controls using allele-specific PCR amplification.ResultsNo differences in the frequency of FcγRIIa genotypes were evident between IPF patients and control subjects. However, significantly impaired pulmonary function at diagnosis was observed in HH compared to RR homozygotes, with evidence of more severe restriction (reduced forced vital capacity (FVC)) and lower diffusing capacity for carbon monoxide (DLCO). Similarly, increased frequency of the H131 allele was observed in patients with severe disease (DLCO < 40% predicted) (0.53 vs. 0.38; p = 0.03). Furthermore, the H131 allele was associated with progressive pulmonary fibrosis as determined by > 10% drop in FVC and/or > 15% fall in DLCO at 12 months after baseline (0.48 vs. 0.33; p = 0.023).ConclusionsThese findings support an association between the FcγRIIa R131H polymorphism and IPF severity and progression, supporting the involvement of immunological mechanisms in IPF pathogenesis.

Copy number variation of FCGR3B is associated with susceptibility to idiopathic pulmonary fibrosis.

Background: Several genes exhibit copy number variation (CNV), including FCGR3B which encodes the IgG receptor FcγRIIIb. Engagement of Fcγ receptors by IgG complexes may contribute to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Objectives: To investigate whether FCGR3B CNV is associated with susceptibility to IPF. Methods: In a case-control study we compared FCGR3B copy number in 142 patients with IPF and in 221 controls by real-time quantitative PCR using CD36 as gene copy control. Results: Significantly increased FCGR3B:CD36 ratio was evident in the IPF cohort compared to controls (p = 0.009). Association of FCGR3B copy number with IPF susceptibility was further confirmed by a likelihood ratio statistical approach (p = 0.003). FCGR3B copy number assignment based on FCGR3B:CD36 ratios revealed significant skewing in the distribution of FCGR3B copy number between IPF patients and controls. In the IPF cohort, there was increased frequency of >2 FCGR3B copies compared to controls (0.30 vs. 0.19; χ2 = 9.27; d.f. 2; p = 0.0097). The presence of >2 FCGR3B copies was associated with higher risk of IPF (p = 0.01, OR: 1.914, 95% CI: 1.17–3.12). Conclusions: These findings support an association of FCGR3B copy number with susceptibility to IPF and propose a novel role for Fcγ receptors in IPF disease pathogenesis.