Professional phagocytes have a vast and sophisticated arsenal of microbicidal features. They are capable of ingesting and destroying invading organisms, and can present microbial antigens on their surface, eliciting acquired immune responses. To survive this hostile response, certain bacterial species have developed evasive strategies that often involve the secretion of effectors to co-opt the cellular machinery of the host. In this Review, we present an overview of the antimicrobial defences of the host cell, with emphasis on macrophages, for which phagocytosis has been studied most extensively. In addition, using Mycobacterium tuberculosis, Listeria monocytogenes, Legionella pneumophila and Coxiella burnetii as examples, we describe some of the evasive strategies used by bacteria.

CD36 participates in macrophage internalization of a variety of particles, and has been implicated in inflammatory responses to many of these ligands. To what extent CD36 cooperates with other receptors in mediating these processes remains unclear. Because CD36 has been shown to cooperate with TLR2, we investigated the roles and interactions of CD36 and TLRs in inflammation and phagocytosis. Using Ab-induced endocytosis of CD36 and phagocytosis of erythrocytes displaying Abs to CD36, we show that selective engagement and internalization of this receptor did not lead to proinflammatory cytokine production by primary human and murine macrophages. In addition, CD36-mediated phagocytosis of Plasmodium falciparum malaria-parasitized erythrocytes (PEs), which contain parasite components that activate TLRs, also failed to induce cytokine secretion from primary macrophages. Furthermore, we demonstrate that CD36-mediated internalization did not require TLR2 or the TLR-signaling molecule IRAK4. However, macrophage pretreatment with TLR agonists markedly stimulated particle uptake via CD36. Similarly, PE uptake was unaffected by TLR deficiency, but in wild-type cells was increased by pretreatment with purified P. falciparum glycosylphosphatidylinositols, which activate TLR2. Our findings indicate that CD36 must cooperate with other receptors such as TLRs to participate in cytokine responses. Although purified P. falciparum components activate TLRs, CD36-mediated internalization of intact PEs is not inflammatory. Further, CD36 mediates internalization of particles, including PEs, independently of TLR signaling, but can functionally cooperate with TLRs to enhance internalization.

Summary CD36 is a versatile receptor known to play a central role in the development of atherosclerosis, the pathogenesis of malaria, and in the removal of apoptotic cells. Remarkably, the short cytosolically-exposed regions of CD36 lack identifiable motifs, which has hampered elucidation of its mode of signaling. Using a combination of phosphoprotein isolation, mass spectrometry, super-resolution imaging and gene silencing we have determined that the receptor induces ligand internalization through a heteromeric complex consisting of CD36, β1 and/or β2 integrins, and the tetraspanins CD9 and/or CD81. This receptor complex serves to link CD36 to the adaptor FcRγ, which bears an immunoreceptor tyrosine activation motif. By coupling to FcRγ, CD36 is able to engage Src-family kinases and Syk, which in turn drives the internalization of CD36 and its bound ligands.

Phagosomes formed by engagement of complement receptors (CR3) are moved within macrophages by PI3K-driven formation of actin “comet tails” on the phagosomal membrane.

Phagocytosis of IgG-opsonized pathogens by Fc␥ receptors requires extensive remodeling of the actin cytoskeleton, a process regulated by the small GTPase Rac. Vav was thought to be the guanine nucleotide exchange factor responsible for the activation of Rac, but recent evidence indicates that Fc␥ receptormediated phagocytosis is unaffected in macrophages lacking all three isoforms of Vav. We therefore tested whether another GEF, DOCK180, participates in Fc␥ receptor-initiated phagocytosis. DOCK180 associates with the adaptor protein Crk, which mediates recruitment of the GEF to sites of tyrosine phosphorylation. CrkII and DOCK180 were found to accumulate at the phagocytic cup. Knockdown of Crk or DOCK180 in murine macrophages using small interfering RNA inhibited phagocytosis of IgG-opsonized particles. Moreover, transfection of dominant negative CrkII prevented both recruitment of DOCK180 and the activation of Rac at the phagocytic cup. This is the first report of a role for either Crk or DOCK180 in Fc␥ receptormediated phagocytosis. The Crk-DOCK180 complex is involved in the clearance of apoptotic cells, which unlike the ingestion of IgG-opsonized particles, is an anti-inflammatory process. The finding that CrkII-DOCK180 is also responsible, at least in part, for the effects of Fc␥ receptors implies that additional, parallel pathways must account for the associated pro-inflammatory effect.

Vasoinhibins are a family of N-terminal prolactin (PRL) fragments that inhibit blood vessel growth, dilation, permeability, and survival. The aspartyl endoprotease cathepsin D is active at acidic pH and can cleave rat PRL to generate vasoinhibins. We investigated whether and where vasoinhibins could be generated by cathepsin D in the adenohypophysis of rats and mice and whether their production could be gender dependent. Vasoinhibins were detected in primary cultures of rat adenohypophyseal cells by Western blot with antibodies directed against the N terminus of PRL but not the C terminus. Ovariectomized, estrogen-treated females show greater levels of adenohypophyseal vasoinhibins than males. Peptide sequencing analysis revealed that the cleaved form of PRL in rat adenohypophyseal extracts contains the PRL N terminus and a second N terminus starting at Ser(149), the reported cleavage site of cathepsin D in rat PRL. In addition, cathepsin D inhibition by pepstatin A reduced vasoinhibin levels in rat adenohypophyseal cell cultures. Confocal and electron microscopy showed the colocalization of cathepsin D and PRL within rat adenohypophyseal cells and secretory granules, and a subcellular fraction of rat adenohypophysis enriched in secretory granules contained cathepsin D activity able to generate vasoinhibins from PRL. Of note, vasoinhibins were absent in the adenohypophysis of mice lacking the cathepsin D gene but not in wild-type mice. These findings show that cathepsin D is the main protease responsible for the generation of adenohypophyseal vasoinhibins and that its action can take place within the secretory granules of lactotrophs.

The amino-terminal 16 kDa fragment of prolactin (16K PRL) promotes the expression of the inducible isoform of nitric oxide synthase (iNOS) accompanied by the production of nitric oxide (NO) by rat pulmonary fibroblasts. The present study was designed to elucidate whether the mechanism by which 16K PRL promotes iNOS expression involves the activation of nuclear factor-kappa B (NF-B), a key transcription factor for iNOS induction. 16K PRL stimulated DNA-binding activity of NF-B in pulmonary fibroblasts as demonstrated by gel shift assays. Likewise, fluorescence immunocytochemistry showed that 16K PRL promotes nuclear translocation of the p65 subunit of NF-B. Finally, treatment with 16K PRL induced the degradation of the NF-B inhibitor B-beta (I B-), and such degradation was prevented by blocking I B-phosphorylation. Altogether, these results show that 16K PRL activates NF-B nuclear translocation via the phosphorylation and degradation of I B-. These findings are consistent with NF-B being part of the signal transduction pathway activated by 16K PRL to induce iNOS expression.

SUMMARY:Diminished oxygen concentration within growing tumors may stimulate neovascularization by inducing both up-regulation of angiogenic factors and down-regulation of antiangiogenic agents. A potentially important molecule in the growth of pituitary adenomas is prolactin (PRL), which can be cleaved by cathepsin-D to yield a 16-kDa form (16K-PRL) with potent antiangiogenic effects. We examined the expression of PRL in cultured GH4C1 pituitary adenoma cells after exposure to hypoxia (0.1% oxygen) for periods of 12 to 36 hours. In contrast to increased expression of the angiogenic factor vascular endothelial growth factor in hypoxic cells, PRL mRNA and levels of intracellular and secreted PRL were significantly reduced under hypoxia. The reduction was not attributable to a general suppression of either transcription or protein synthesis. Although 16K-PRL was not evident in conditioned medium at physiologic pH, lowering the pH to mimic the acidic tumor microenvironment resulted in generation of 16K-PRL, which was sharply reduced in medium drawn from hypoxic cells. Production of 16K-PRL was blocked by the cathepsin-D inhibitor pepstatin-A, and the reduced 16K-PRL formation in hypoxic-conditioned medium correlated with a decrease in secretion of cathepsin-D and its precursor, procathepsin-D. Thus, hypoxia acts upon GH4C1 cells to increase vascular endothelial growth factor expression, decrease PRL synthesis, and suppress conversion of PRL to 16K-PRL via inhibition of cathepsin-D proteolysis. These mechanisms may act in concert to stimulate angiogenesis in prolactinomas. (Lab Invest 2003, 83:1627-1636.