Identifying the functional part of heparin-binding protein (HBP) as a monocyte stimulator and the novel role of monocytes as HBP producers.

Heparin-binding protein (HBP), an evolutionary ancient and biologically highly important molecule in inflammation, is an inactive serine protease due to mutations in the catalytic triad. The histidine (position 41) in the conserved sequence TAAHC is mutated to serine and this sequence (TAASC) plays a crucial role when HBP binds to monocytes. We synthesized a 20-44 HBP peptide, cyclicized by a sulphur bridge, which encompasses this amino acid and functions as full-length HBP. Using a human monocyte cell line, we have shown that lipopolysaccharide (LPS)-triggered secretion of IL-6 is enhanced up to 10-fold when full-length HBP or the peptide are present in low-to-moderate concentrations. A monoclonal antibody neutralizing HBP also neutralizes the peptide, indicating that the ligand for the HBP receptor is located near serine in position 41 on the HBP surface. A 'back mutated' 20-44 peptide (serine→histidine) has some, but not significant, stimulatory effect on monocytes. Normally, HBP production and release is ascribed to neutrophil granulocytes, but here we find that also monocytes secrete HBP when stimulated with LPS. Furthermore, a small amount of HBP can be demonstrated when monocytes are incubated in medium alone. Our efforts to identify a suggested HBP receptor on monocytes has failed so far.

Neutrophil-derived heparin-binding protein (HBP/CAP37) deposited on endothelium enhances monocyte arrest under flow conditions.

In acute inflammation, infiltration of neutrophils often precedes a second phase of monocyte invasion, and data in the literature suggest that neutrophils may directly stimulate mobilization of monocytes via neutrophil granule proteins. In this study, we present a role for neutrophil-derived heparin-binding protein (HBP) in monocyte arrest on endothelium. Adhesion of neutrophils to bovine aorta endothelial cells (ECs) or HUVEC-triggered secretion of HBP and binding of the protein to the EC surface. Blockade of neutrophil adhesion by treatment with a mAb to CD18 greatly reduced accumulation of HBP. In a flow chamber model, immobilized recombinant HBP induced arrest of human monocytes or monocytic Mono Mac 6 (MM6) cells to activated EC or plates coated with recombinant adhesion molecules (E-selectin, P-selectin, VCAM-1). However, immobilized recombinant HBP did not influence arrest of neutrophils or lymphocytes. Treatment of MM6 cells with recombinant HBP evoked a rapid and clear-cut increase in cytosolic free Ca(2+) that was found to be critical for the HBP-induced monocyte arrest inasmuch as pretreatment with the intracellular calcium chelating agent BAPTA-AM abolished the evoked increase in adhesion. Thus, secretion of a neutrophil granule protein, accumulating on the EC surface and promoting arrest of monocytes, could contribute to the recruitment of monocytes at inflammatory loci.

Effects of heparin-binding protein (CAP37/azurocidin) in a porcine model of Actinobacillus pleuropneumoniae-induced pneumonia.

Heparin-binding protein (HBP; CAP37/azurocidin) is secreted from neutrophil leukocytes early during inflammation and plays a central role in early capillary leakage and extravasation of neutrophils. Furthermore, HBP is chemotactic towards monocytes and lymphocytes and protects against stress-induced apoptosis, e.g. induced by oxygen radicals released during inflammation. Thus, administration of HBP as an adjunct to antibiotics increased survival of mice with peritonitis. In the present study, the effects of recombinant HBP as an adjunct to standard antibiotics were examined in a porcine model of Actinobacillus pleuropneumoniae-induced pneumonia. Beneficial and possible adverse effects of HBP were evaluated clinically and pathologically as well as by measurement of clinical chemical variables and markers of inflammation (interleukin-6 and C-reactive protein) and oxidative stress (ascorbic acid and alpha-tocopherol). Pigs receiving HBP (0.55 mgkg-1, n=11) as a 6-hourly subcutaneous infusion starting 1-h post-infection had a faster decrease in rectal temperature compared to control animals receiving a carrier-infusion (n=11), with a significant lower temperature at 32 h post-infection (p<0.05). This difference was, however, transient and the temperature curves had a similar course from 38 h and onwards. The transient effect of HBP might be explained by the dosage regimen that was used. No signs of adverse effects of the HBP-infusion were observed. More studies are needed to elucidate of the effects of HBP further and to optimise the dosage regimen for further improvement the efficacy and safety.

M protein, a classical bacterial virulence determinant, forms complexes with fibrinogen that induce vascular leakage.

Increased vascular permeability is a key feature of inflammatory conditions. In severe infections, leakage of plasma from the vasculature induces a life-threatening hypotension. Streptococcus pyogenes, a major human bacterial pathogen, causes a toxic shock syndrome (STSS) characterized by excessive plasma leakage and multi-organ failure. Here we find that M protein, released from the streptococcal surface, forms complexes with fibrinogen, which by binding to beta2 integrins of neutrophils, activate these cells. As a result, neutrophils release heparin binding protein, an inflammatory mediator inducing vascular leakage. In mice, injection of M protein or subcutaneous infection with S. pyogenes causes severe pulmonary damage characterized by leakage of plasma and blood cells. These lesions were prevented by treatment with a beta2 integrin antagonist. In addition, M protein/fibrinogen complexes were identified in tissue biopsies from a patient with necrotizing fasciitis and STSS, further underlining the pathogenic significance of such complexes in severe streptococcal infections.

Secretion of heparin-binding protein from human neutrophils is determined by its localization in azurophilic granules and secretory vesicles.

Human neutrophils have an important role in host defense against microbial infection. At different stages of an infectious process, neutrophils progressively up-regulate receptors and release various effector molecules. These are stored in several distinct types of granules with varying propensity to be secreted. Heparin-binding protein (HBP), also known as CAP37 or azurocidin, is a multifunctional, inactive serine-protease homologue. The present work shows that HBP is released from neutrophils on stimulation with secretagogues that do not trigger the secretion of azurophilic granule content. Therefore, the subcellular localization of HBP was investigated in more detail. Immunofluorescence microscopy revealed that HBP was localized close to the plasma membrane. Further analysis by fractionation of postnuclear supernatants from cavitated neutrophils showed that HBP is stored in azurophilic granules and secretory vesicles but that it is also detected to a minor extent in the plasma membrane. These findings were confirmed by immunoelectron microscopy showing that HBP colocalized with marker proteins of azurophilic granules and secretory vesicles. The presence of HBP in secretory vesicles possibly depends on the stage of cell differentiation, since the promyelocytic cell line HL-60 contains less HBP than mature neutrophils, stored exclusively in the less easily mobilized azurophilic granules. Our findings suggest that HBP can be synthesized or targeted to easily mobilized compartments at a late stage of neutrophil maturation. The ability of neutrophils to secrete HBP from secretory vesicles may be important for proinflammatory functions of this protein, such as the alteration of vascular permeability.