CD44, a transmembrane adhesion molecule involved in binding and metabolism of hyaluronan, has additional functions in inflammatory and immune responses, contributing to the ingestion and clearance of particles and apoptotic cells. Our goal was to determine the specific role of CD44 in phagocytosis and whether it functions as a primary or accessory phagocytic receptor. Using hyaluronan-coated beads and erythrocytes coated with anti-CD44 antibodies as the phagocytic prey, we determined that CD44 mediates efficient phagocytosis in primary murine peritoneal macrophages and in the murine macrophage cell line RAW 264.7. In RAW cells, the phagocytic index for anti-CD44-coated erythrocytes was 25 ؎ 3 (mean ؎ SEM) compared with less than 1 for erythrocytes coated with isotype-matched control antibodies. Uptake of anti-CD44-coated erythrocytes was abrogated by pretreatment with a blocking antibody to CD44 and was absent in primary cultures of CD44-deficient murine macrophages. Down-regulation of Fc receptors by aggregated IgG-induced internalization, which blocks uptake of IgG-coated particles, had no effect on CD44-mediated particle engulfment. Using a combination of immunoprecipitation, pharmacologic inhibition, and genetic deletion, we determined that CD44-mediated phagocytosis involves Syk, Rac1, and phosphatidylinositol 3-kinase and induced activation of the phagocyte oxidase. We conclude that CD44 is a competent phagocytic receptor that efficiently mediates internalization of large particles. IntroductionThe transmembrane adhesion molecule CD44, known to be involved in binding, endocytosis, and metabolism of hyaluronan, 1-3 also has additional functions in innate and adaptive immunity. CD44 encompasses a heterogeneous family of receptors with isoforms ranging from 80 to 200 kDa that are encoded by a single gene composed of 19 exons. 4 This extensive heterogeneity of CD44 is attributable to a combination of alternate splicing 4 and extensive N-and O-glycosylation. [5][6][7][8] Purported ligands of CD44 include growth factors such as basic fibroblast growth factor and heparin-binding epidermal growth factor, 5,6 fibronectin, 9 collagen, 10 fibrin, 11,12 the proteoglycan serglycin found in lymphocytes and mast-cell granules, 13 osteopontin, 14 and other CD44 molecules on vicinal cells. 15 Diverse functions have been attributed to CD44, including involvement in cellular adhesion and migration, lymphocyte and monocyte homing, activation and proliferation, cytocidal activity of natural killer cells, and tumor metastasis. 16 Observations also point to a role for CD44 in binding, ingestion (phagocytosis), and clearance of apoptotic cells 17 and microbial pathogens. 18 With respect to clearance of apoptotic cells, direct antibody ligation of CD44 on macrophages 19 or on airway epithelial cells 20 enhanced subsequent uptake of apoptotic neutrophils and eosinophils, respectively, suggesting a costimulatory function for the receptor. However, ensuing reports have suggested that some of these effects may be attributed to the Fc...

Diverse receptors, including Fc␥ receptors and ␤ 2 integrins (complement receptor-3 [CR3], CD11b/CD18), have been implicated in phagocytosis, but their distinct roles and interactions with other receptors in particle engulfment are not well defined. CD44, a transmembrane adhesion molecule involved in binding and metabolism of hyaluronan, may have additional functions in regulation of inflammation and phagocytosis. We have recently reported that CD44 is a fully competent phagocytic receptor that is able to trigger ingestion of large particles by macrophages. Here, we investigated the role of coreceptors and intracellular signaling pathways in modulation of CD44-mediated phagocytosis. Using biotinylated erythrocytes coated with specific antibodies (anti-CD44-coated erythrocytes [Ebabs]) as the phagocytic prey, we determined that CD44-mediated phagocytosis is reduced by 45% by a blocking CD11b antibody. Further, CD44-mediated phagocytosis was substantially (42%) reduced in CD18-null mice. Immunofluorescence microscopy revealed that CD11b is recruited to the phagocytic cup.The mechanism of integrin activation and mobilization involved activation of the GTPase Rap1. CD44-mediated phagocytosis was also sensitive to the extracellular concentration of the divalent cation Mg 2؉ but not Ca 2؉ . In addition, buffering of intracellular Ca 2؉ did not affect CD44-mediated phagocytosis. Taken together, these data suggest that CD44 stimulation induces inside-out activation of CR3 through the GTPase Rap1. IntroductionComplement receptor-3 (CR3, Mac1, CD11b/CD18) is a member of the ␤ 2 integrin family of adhesion receptors, which includes CR4 (CD11c/CD18) and LFA-1 (CD11a/CD18). These heterodimeric transmembrane receptors, composed of an ␣ chain (CD11a, CD11b, or CD11c) and the common ␤ 2 chain (CD18), are expressed exclusively on cells of hematopoietic origin. CR3 is multifunctional and is known to be involved in leukocyte adhesion, diapedesis, and phagocytosis. 1 Unlike phagocytosis mediated by Fc receptors, CR3-mediated phagocytosis is regulated by the activation state of the receptor. On quiescent (resting) leukocytes and monocytes, CR3 is capable of binding but not ingesting iC3b-coated phagocytic prey. In contrast, prior activation of leukocytes by diverse agents such as phorbol ester 2 and extracellular matrix proteins such as fibrinogen [3][4][5] activates CR3 and renders it capable of particle ingestion (phagocytosis).While many studies of phagocytosis have used artificial particles such as erythrocytes or latex beads opsonized with monospecific ligands as phagocytic prey, microbial pathogens such as bacteria and fungi express multiple ligands on their cell surfaces that can be recognized by phagocytes. 6,7 The simultaneous engagement of multiple receptors on the host cell by the pathogen ensures that diverse cellular signaling pathways are activated, and the resultant cellular response will therefore reflect a composite of the effects of activation of the different receptors. Examples of cross-talk between receptors in...

Proteinases can influence lung inflammation by various mechanisms, including via cleavage and activation of protease-activated receptors (PAR) such as PAR2. In addition, proteinases such as neutrophil and/or Pseudomonas-derived elastase can disarm PAR2 resulting in loss of PAR2 signaling. Currently, the role of PAR2 in host defense against bacterial infection is not known. Using a murine model of acute Pseudomonas aeruginosa pneumonia, we examined differences in the pulmonary inflammatory response between wild-type and PAR2(-/-) mice. Compared with wild-type mice, PAR2(-/-) mice displayed more severe lung inflammation and injury in response to P. aeruginosa infection as indicated by higher bronchoalveolar lavage fluid neutrophil numbers, protein concentration, and TNF-alpha levels. By contrast, IFN-gamma levels were markedly reduced in PAR2(-/-) compared with wild-type mice. Importantly, clearance of P. aeruginosa was diminished in PAR2(-/-) mice. In vitro testing revealed that PAR2(-/-) neutrophils killed significantly less bacteria than wild-type murine neutrophils. Further, both neutrophils and macrophages from PAR2(-/-) mice displayed significantly reduced phagocytic efficiency compared with wild-type phagocytes. Stimulation of PAR2 on macrophages using a PAR2-activating peptide resulted in enhanced phagocytosis directly implicating PAR2 signaling in the phagocytic process. We conclude that genetic deletion of PAR2 is associated with decreased clearance of P. aeruginosa. Our data suggest that a deficiency in IFN-gamma production and impaired bacterial phagocytosis are two potential mechanisms responsible for this defect.

Pulmonary infection is the dominant clinical feature of cystic fibrosis (CF), but the basis for this susceptibility remains incompletely understood. One hypothesis is that CF airway surface liquid (ASL) is abnormal and interferes with neutrophil function. To study this possibility, we developed an in vitro system in which we collected ASL from primary cultures of normal and CF airway epithelial cells. Microbial killing was less efficient when bacteria were incubated with neutrophils in the presence of ASL from CF epithelia compared with normal ASL. Antimicrobial functions of human neutrophils were assessed in ASL from CF and normal epithelia using a combination of quantitative bacterial culture, flow cytometry, and microfluorescence imaging. The results of these assays of neutrophil function were indistinguishable in CF and normal ASL. In contrast, the direct bactericidal activity of ASL to Escherichia coli and to clinical isolates of Staphylococcus aureus and Pseudomonas aeruginosa was substantially less in CF than in normal ASL, even when highly diluted in media of identical ionic strength. Together, these observations indicate that the antimicrobial properties of ASL in CF are compromised in a manner independent of ionic strength of the ASL, and that this effect is not mediated through a direct effect of the ASL on phagocyte function.