Platelet-derived chemokines: pathophysiology and therapeutic aspects.

The identification of chemokines in blood platelets has strengthened our view of these cells as participants in immune host defense. Platelet chemokines representing prestored and rapidly releasable proteins may play a major role as first-line inflammatory mediators. This is evident from their capability to recruit early inflammatory cells such as neutrophil granulocytes and monocytes and even to exhibit direct antimicrobial activity. However, insight is growing that platelet chemokines may be also long-term regulators, e.g., by activating T lymphocytes, by modulating the formation of endothelium and even thrombocytopoiesis itself. This review deals with the individual and cooperative functionality of platelet chemokines, as well as their potential as a basis for therapeutic intervention in the pathology of inflammation, infection, allergy and tumors. Within this context, therapeutic strategies based on the use of antibodies, modified chemokines, chemokine-binding proteins and chemokine receptor antagonists as well as first clinical studies will be addressed.

The CXC chemokine NAP-2 mediates differential heterologous desensitization of neutrophil effector functions elicited by platelet-activating factor.

During early inflammation, the chemoattractants neutrophil-activating peptide-2 (NAP-2), platelet-activating factor (PAF), and complement component C5a are rapidly generated within the vasculature and potently induce effector functions in neutrophils, such as chemotaxis and degranulation. We investigated whether these mediators would cross-desensitize each other's activities on isolated neutrophils. We demonstrate that NAP-2 and C5a desensitize degranulation of neutrophils in response to PAF. However, whereas C5a-mediated desensitization correlated with the downregulation of PAF binding sites on neutrophils, NAP-2 did not regulate PAF receptors, nor did it modulate their calcium signaling. In further contrast to C5a, which desensitized PAF-induced neutrophil chemotaxis, NAP-2 did not affect the chemotatic response to PAF. These observations indicate that NAP-2 mediates selective desensitization of PAF-induced neutrophil degranulation by a mechanism downstream from PAF receptors, still allowing migration toward PAF. Thus, a role for NAP-2 may be to limit PAF-dependent vascular tissue damage by preventing degranulation of neutrophils without affecting their migration into the inflamed tissue.

Control of mycobacterial replication in human macrophages: roles of extracellular signal-regulated kinases 1 and 2 and p38 mitogen-activated protein kinase pathways.

Intracellular persistence of mycobacteria may result from an intricate balance between bacterial replication and signaling events leading to antimicrobial macrophage activities. Using human monocyte-derived macrophages, we investigated the relevance of mitogen-activated protein kinase activation for the growth control of Mycobacterium avium isolates differing in their abilities to multiply intracellularly. The highly replicative smooth transparent morphotype of M. avium strain 2151 induced significantly less p38 and extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation than the smooth opaque morphotype of the same strain, which was gradually eliminated from macrophage cultures. Inhibition of the p38 pathway by highly specific inhibitors did not significantly affect mycobacterial replication within macrophages, regardless of the in vitro virulence of the M. avium strain. However, repression of the ERK1/2 pathway further enhanced intracellular growth of highly replicative M. avium strains, although it did not increase survival of the poorly replicating M. avium isolate. Inhibition of the ERK1/2 pathway resulted in decreased tumor necrosis alpha (TNF-alpha) secretion irrespective of the virulence of the M. avium isolate used for infection, revealing that TNF-alpha could have been only partially responsible for the control of intracellular M. avium growth. In conclusion, ERK1/2- and TNF-alpha-independent pathways are sufficient to limit intramacrophage growth of less-virulent M. avium strains, but early ERK1/2 activation in infected macrophages is critically involved in controlling the growth of highly replicative M. avium strains.

Increased IL-10 production during spontaneous apoptosis of monocytes.

Monocytes/macrophages undergo apoptosis and are in contact with apoptotic cells both in vitro and in vivo. The data show that monocytes undergoing spontaneous apoptosis in vitro change their cytokine production profile. We demonstrate that the lipopolysaccharide (LPS)-induced production of interleukin-10 (IL-10) is up-regulated, while production of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) is either not affected or reduced. These differences seen both at the protein and mRNA level directly correlate with the appearance of apoptotic cells in the culture. Flow cytometry analysis using double staining, surface with annexin V and intracellular with anti-IL-10, suggested that annexin V-negative monocytes are the predominant source of IL-10. Analysis of sorted populations of monocytes indicated that the increase in IL-10 synthesis appears to result from direct interactions between non-apoptotic and apoptotic cells at the time of stimulation. Also non-apoptotic, freshly isolated monocytes produced more IL-10 upon stimulation with LPS, Staphylococcus aureus or zymosan when apoptotic neutrophils were added to the culture. In contrast, monocyte-derived macrophages did not produce more IL-10 in the presence of apoptotic neutrophils. Finally, we found that the presence of apoptotic monocytes in the culture may influence specific immune responses. The data show that in the presence of annexin V-positive monocytes CD4-positive memory T cells produce less IFN-gamma upon stimulation with purified protein derivative of tuberculin, which could be partially reversed by anti-IL-10 neutralizing antibodies. We conclude that these findings might illustrate the mechanisms operating within an inflammatory site and play an important immunoregulatory role during the resolution of inflammation and specific immune responses.

Platelet-derived chemokines CXC chemokine ligand (CXCL)7, connective tissue-activating peptide III, and CXCL4 differentially affect and cross-regulate neutrophil adhesion and transendothelial migration.

In this study, we have examined the major platelet-derived CXC chemokines connective tissue-activating peptide III (CTAP-III), its truncation product neutrophil-activating peptide 2 (CXC chemokine ligand 7 (CXCL7)), as well as the structurally related platelet factor 4 (CXCL4) for their impact on neutrophil adhesion to and transmigration through unstimulated vascular endothelium. Using monolayers of cultured HUVEC, we found all three chemokines to promote neutrophil adhesion, while only CXCL7 induced transmigration. Induction of cell adhesion following exposure to CTAP-III, a molecule to date described to lack neutrophil-stimulating capacity, depended on proteolytical conversion of the inactive chemokine into CXCL7 by neutrophils. This was evident from experiments in which inhibition of the CTAP-III-processing protease and simultaneous blockade of the CXCL7 high affinity receptor CXCR-2 led to complete abrogation of CTAP-III-mediated neutrophil adhesion. CXCL4 at substimulatory dosages modulated CTAP-III- as well as CXCL7-induced adhesion. Although cell adhesion following exposure to CTAP-III was drastically reduced, CXCL7-mediated adhesion underwent significant enhancement. Transendothelial migration of neutrophils in response to CXCL7 or IL-8 (CXCL8) was subject to modulation by CTAP-III, but not CXCL4, as seen by drastic desensitization of the migratory response of neutrophils pre-exposed to CTAP-III, which was paralleled by selective down-modulation of CXCR-2. Altogether our results demonstrate that there exist multiple interactions between platelet-derived chemokines in the regulation of neutrophil adhesion and transendothelial migration.

Platelet factor 4 inhibits proliferation and cytokine release of activated human T cells.

Platelet factor 4 (PF-4), a platelet-derived CXC chemokine, has been shown to induce the differentiation of monocytes into a subset of macrophages that lack the expression of HLA-DR Ag. This suggests a potential role for PF-4 in the modulation of monocyte-dependent T cell activation. Using an Ag-specific stimulation model in which T cells were cocultured with monocytes in the presence of recall Ags, we could show that under these conditions PF-4-treatment caused a strong decrease of T cell proliferation as well as of IFN-gamma release. However, inhibition of T cell functions such as proliferation, IL-2 release, and IL-2 mRNA production did also occur when isolated T cells were activated in the absence of monocytes with immobilized Abs directed against CD3 in combination with cross-linked anti-CD28 Abs. The effect could be reversed when low concentrations of exogenous IL-2 instead of anti-CD28 were used as a costimulus in combination with anti-CD3 Abs. Further evidence for direct modulation of T cell function by PF-4 was obtained by the detection of specific binding sites for the chemokine on the surface of these cells. Taken together, our results show that specific binding of PF-4, resulting in the down-regulation of the IL-2-release correlates with the inhibition of functions in activated T cells.