IntroductionImmunity to infections depends on the successful integration of innate and adaptive defense strategies. 1 Cells of the innate immune system, such as macrophages and dendritic cells, recognize pathogen-associated molecular patterns shared by many microbes but not found in higher eukaryotes, via members of the Toll-like receptor (TLR) family. 2,3 TLR-dependent signaling pathways can directly induce macrophage antimicrobial programs but also initiate inflammatory cell recruitment and help prime cells of the adaptive immune system to amplify bactericidal effector mechanisms. Experimental infections with microorganisms have been used successfully to uncover the intricacies governing the interplay between innate and adaptive immunity. 4,5 For example, cell-wall components of Mycobacterium tuberculosis, the causative organism of tuberculosis, critically depend on TLR2 and TLR4 to induce secretion of the proinflammatory cytokines tumor necrosis factor alpha (TNF-␣) and interleukin (IL-12, necessary for differentiating T-helper 1 cells). 6 Subsequently, mycobacteria-primed T cells secrete interferon gamma (IFN-␥) as a critical macrophageactivating agent. Eradication of mycobacteria is achieved only when both arms of the immune system are fine-tuned for full antimicrobial potency.Functionally, the Toll-mediated induction of antimicrobial effector systems is highly conserved between Drosophila melanogaster (D melanogaster) and Homo sapiens. 7,8 Another example for human evolutionarily ancient effector mechanisms is granulysin (a granule-stored bactericidal molecule), which is homologous to the amoebapores of Entamoeba histolytica. 9,10 Thus, there is ample precedence for evolutionarily conserved signatures governing individual facets of the immune response. To uncover novel regulatory pathways in innate responses to infection, we performed a microarray-based gene-expression screen with human macrophages infected with mycobacteria or conserved bacterial structures. We found mRNA for WNT5A, a homolog of Wingless in Drosophila species, to be consistently up-regulated in response to all stimuli.Wingless was originally characterized as a segment polarity gene in D melanogaster, which is essential in embryonic segmentation and patterning (reviewed in Klingensmith and Nusse 11 ). Various homologs of the Wingless protein, termed WNT, are involved in embryonic development of nonvertebrates and vertebrates, 12,13 where WNT signaling determines cell motility, differentiation, and apoptosis. 14 In mammalian hematopoiesis, WNT signaling is essential for stem-cell homeostasis 15 and lymphocyte differentiation. 16,17 Most recently, one member of the WNT family of proteins, WntD, was shown to be involved in regulating antibacterial defenses in Drosophila. 18 To date, however, WNT Center Borstel, Germany), phorbol-12-myristate13-acetate (PMA), calcium ionophore A23187 (both Sigma Aldrich, Taufkirchen, Germany), and phytohemagglutinin (PHA) were used for stimulation. To rule out the presence of lipopolysaccharides (LPSs) in the...

Antibacterial proteins are components of the innate immune system found in many organisms and produced by a variety of cell types. Human blood platelets contain a number of antibacterial proteins in their ␣-granules that are released upon thrombin activation. The present study was designed to purify these proteins obtained from human platelets and to characterize them chemically and biologically. Two antibacterial proteins were purified from platelet granules in a two-step protocol using cation exchange chromatography and continuous acid urea polyacrylamide gel electrophoresis and were designated thrombocidin (TC)-1 and TC-2. Characterization of these proteins using mass spectrometry and Nterminal sequencing revealed that TC-1 and TC-2 are variants of the CXC chemokines neutrophil-activating peptide-2 and connective tissue-activating peptide-III, respectively. TC-1 and TC-2 differ from these chemokines by a C-terminal truncation of 2 amino acids. Both TCs, but not neutrophil-activating peptide-2 and connective tissue-activating peptide-III, were bactericidal for Bacillus subtilis, Escherichia coli, Staphylococcus aureus, and Lactococcus lactis and fungicidal for Cryptococcus neoformans. Killing of B. subtilis by either TC appeared to be very rapid. Because TCs were unable to dissipate the membrane potential of L. lactis, the mechanism of TC-mediated killing most probably does not involve pore formation.

Unstimulated monocytes rapidly undergo physiological changes resulting in programmed cell death (apoptosis) while stimuli promoting differentiation of these cells into macrophages were shown to inhibit apoptotic processes. In the present study, we report that the platelet-derived -chemokine platelet factor 4 (PF4) induces the differentiation of monocytes into macrophages, as is evident from morphological changes as well as from the up-regulation of differentiation markers (carboxypeptidase M/MAX1 and CD71). Significant alterations of the phenotype were observed after 72 hours of culture in the presence of the chemokine and required a minimal concentration of 625 nmol/L PF4. PF4-induced macrophages were characterized by a lack of HLA-DR antigen on their surface but showed a strong increase in the expression of the CD28 ligand B7-2. Furthermore, PF4 stimulation prevented monocytes from undergoing spontaneous apoptosis during 72 hours of culture as determined in an annexin-V–binding assay. Although PF4 induced the secretion of relevant amounts of TNF-, neutralizing antibodies directed against TNF- or granulocyte-macrophage colony–stimulating factor (GM-CSF) did not revert PF4-induced rescue from programmed cell death, suggesting that PF4 exerts its antiapoptotic effects in a TNF-– or GM-CSF–independent fashion. On the basis of these results, we propose a novel role for PF4 in the control of monocyte differentiation during an inflammatory process in vivo.

The recruitment of neutrophil granulocytes to sites of tissue injury is one of the earliest events during host defense. Several chemotactic cytokines belonging to the CXC subfamily of chemokines are thought to be implicated in this kind of response. Especially those CXC chemokines that are stored in blood platelets and become immediately released upon activation are likely to dominate neutrophil-dependent host defense at the onset of inflammation. The major platelet-derived CXC chemokines are the ␤-thromboglobulins and platelet factor 4 (PF-4), which are both released into the blood at micromolar concentrations. The availability as well as the functional activity of these mediators appear to be subject to tight control by diverse regulatory mechanisms. These include proteolytic processing of chemokine precursors, oligomer formation, and the differential usage of neutrophil-expressed receptors. Herein we review our work on early neutrophil regulation by PF-4, the ␤-thromboglobulin neutrophil-activating peptide 2 (NAP-2) and its major precursor connective tissueactivating peptide III (CTAP-III). We moreover propose a model to assess the contribution by either of these chemokines to coordinated recruitment and activation of neutrophils in response to acute tissue injury. J. Leukoc. Biol. 67: 471-478; 2000.

Although platelet factor 4 (PF-4) and the beta-thromboglobulin (beta-TG) proteins represent the first chemokines to be discovered, their functional roles in host defense became clear only recently. Residing in platelets as storage proteins and becoming released into the blood at very high concentrations, these mediators appear to fulfill different and complementary tasks as first-line mediators in the recruitment and activation of leukocytes, as well in the regulation of tissue repair. Whereas both proteins are structurally closely related members of the CXC chemokine subfamily, they are subject to quite dissimilar regulatory mechanisms controlling their generation and their spectrum of biological activities. Thus, proteolytic processing of inactive precursors plays a decisive role in whether the beta-TG proteins will act as stimulatory or inhibitory agents in neutrophil activation via the G protein-coupled receptors CXCR-1 and 2. PF-4, existing as a single molecular form, is largely resistant to proteolytic modification, but its interaction with an unusual receptor(s) on leukocytes (a proteoglycan) appears to depend on its oligomeric state. There is growing evidence that both chemokines may interfere with each other at various regulatory levels to promote coordinated cell activation. Moreover, recent findings suggest novel and unexpected activities for these chemokines, which may extend our view on early host defense.