Meytham Majeed

Activation of cAMP‐dependent protein kinase is necessary for actin rearrangements in human neutrophils during phagocytosis

We have investigated the role of cAMP and cAMP‐dependent protein kinase (cAPK) in neutrophil phagocytosis. Inhibition of cAPK with H‐89 reduced complement‐ and IgG‐dependent phagocytosis to 83 and 46%, respectively. Fluorescence intensity measurements of phalloidin‐stained actin in neutrophils showed a reduced amount of filamentous actin (F‐actin) in pseudopods and around the phagosome in cells treated with H‐89 or cAMP‐elevating agents (forskolin and rolipram). The amount of phosphotyrosine‐containing proteins was also reduced in pseudopods and around the phagosome. Taken together, the data show that cAMP/cAPK regulates F‐actin reorganization during receptor‐mediated phagocytosis, particularly triggered by IgG‐FcR interaction. Our results support the hypothesis that active subcortical reorganization of F‐actin is a prerequisite for FcR‐mediated phagocytosis, but is less important during CR3‐mediated ingestion. J. Leukoc. Biol. 67: 520–528; 2000.

ROLE OF ANNEXINS IN ENDOCYTOSIS OF ANTIGENS IN IMMATURE HUMAN DENDRITIC CELLS

We have evaluated the uptake of a soluble protein antigen, denitrophenylated human serum albumin (DNP‐HSA), and two different intracellular bacteria; Chlamydia trachomatis serovar L2 and Mycobacterium tuberculosis strain H37Ra, by immature human dendritic cells. These were generated by culturing progenitor cells from blood in the presence of cytokines (granulocyte–macrophage colony‐stimulating factor and interleukin‐4). Dendritic cells play a crucial part in antigen presentation for the induction of T‐cell‐dependent immune responses in various tissues. Recently, macropinocytic and phagocytic activity has been shown for immature dendritic cells of mouse, rat and human origin. In the present study, macropinocytosis characterized the uptake of the soluble protein‐antigen DNP‐HSA, whereas the C. trachomatis were ingested via receptor‐mediated endocytosis in coated pits, and opsonized M. tuberculosis via phagocytosis. To follow the intracellular routes of the antigens, their positions were compared with the localization of annexins, a family of Ca2+‐and phospholipid‐binding proteins, involved in membrane fusion, aggregation and transport of different vesicles. To elucidate further the intracellular pathway of the antigens, two other proteins, lysosome‐associated membrane protein‐1 (LAMP‐1) and cathepsin D, were labelled. They are known to colocalize with major histocompatibility complex class II compartments in the immature dendritic cells. We observed a distinct translocation of annexin V to DNP‐HSA containing endosomes, and annexin III to vesicles with C. trachomatis. Furthermore, annexin III, IV and V redistributed to phagosomes with M. tuberculosis. Both LAMP‐1 and cathepsin D colocalized with DNP‐HSA endosomes, and with phagosomes with M. tuberculosis. Thus, immature human dendritic cells have the capacity to phagocytose. Moreover, the handling of these antigens by dendritic cells may represent three distinct intracellular pathways, albeit some properties and compartments are shared.

Chlamydia pneumoniae Binds to Platelets and Triggers P-Selectin Expression and Aggregation: A Causal Role in Cardiovascular Disease?

Objective—Evidence linking Chlamydia pneumoniae to atherosclerotic cardiovascular disease is expanding. Platelets are considered to play an essential role in cardiovascular diseases; however, so far platelets have not been associated with an infectious cause of atherosclerosis. This study aims to clarify the interaction between C pneumoniae and platelets and possibly present a novel mechanism in the pathogenesis of atherosclerosis. Methods and Results—The effects of C pneumoniae on platelet aggregation and secretion were assessed with lumiaggregometry, and the ability of C pneumoniae to bind to platelets and stimulate expression of P-selectin was analyzed with flow cytometry. We found that C pneumoniae, at a chlamydia:platelet ratio of 1:15, adheres to platelets and triggers P-selectin expression after 1 minute and causes an extensive aggregation and ATP secretion after 20 minutes of incubation. Inhibition of glycoprotein IIb/IIIa with Arg-Gly-Asp-Ser or abciximab markedly reduced C pneumoniae-induced platelet aggregation. Exposure of C pneumoniae to polymyxin B, but not elevated temperature, abolished the stimulatory effects on platelet activation, suggesting that chlamydial lipopolysaccharide has an active role. In contrast, other tested bacteria had no or only moderate effects on platelet functions. Conclusion—Our findings demonstrate a new concept of how C pneumoniae activates platelets and thereby may cause atherosclerosis and thrombotic vascular occlusion.

Binding of Chlamydia trachomatis Serovar L2 to Collagen Types I and IV, Fibronectin, Heparan Sulphate, Laminin and Vitronectin

Chlamydia trachomatis serovar L2 elementary bodies were tested for binding to 125I-labelled components of the extracellular matrix. C. trachomatis bound high levels of collagen type I and heparan sulphate, whereas the binding to collagen type IV, fibronectin, laminin and vitronectin was substantially less. Preliminary characterization showed that chlamydial surface proteins are essential for binding to occur. Our results suggest that a selective affinity for C. trachomatis to a limited number of components of the extracellular matrix may have important implications mediating attachment to host cell surfaces and localization of chlamydiae in other tissues, such as joints.

Mobilization of annexin V during the uptake of DNP-albumin by human dendritic cells.

Dendritic cells play a crucial role in antigen presentation in various tissues. The endocytic capacity of these cells has been regarded as minimal, but recent work on dendritic cells from mouse spleen has disclosed that the fluid‐phase traffic through late endosomes is as active in dendritic cells as in other antigen‐presenting cell types. We show that cultured human dendritic cells express the annexins I, III, IV, V and VI, as detected by immunofluorescence staining. The annexins are cytosolic Ca2+‐dependent proteins with the ability to promote vesicle aggregation and membrane fusion through their capacity to bind to membrane phospholipids. Annexin I and VI appeared to outline the cytoskeleton and the plasma membrane in cultured human dendritic cells. Studies using confocal laser scanning microscopy showed that during the endocytosis of fluorescent dinitrophenyl‐conjugated albumin by dendritic cells, there was a redistribution of annexin V which was found to colocalize with vesicles containing dinitrophenyl‐FITC‐conjugated albumin.

Annexin Expression in Human Dendritic Cells

Dendritic cells are the most potent antigen-presenting cells for the induction of primary antigen-specific T-cell responses in vitro and in situ (1). The first step in antigen-presentation is endocytosis of the antigen followed by antigen degradation to antigen peptides, binding to the major histocompatibility complex molecule and transport to the cell surface or exocytosis. The endocytotic capacity of dendritic cells has been regarded as minimal, but recent work on dendritic cells from mouse spleen has shown that the fluid phase traffic through late endosomes is as active in spleen dendritic cells as in other types of antigen-presenting cells (2). To further investigate the antigen endocytosis and exocytosis pathway in human peripheral blood dendritic cells we mapped the distribution of annexins in the dendritic cell cytoplasm.