Hans Flodgaard

Heparin-binding protein (HBP/CAP37): a missing link in neutrophil-evoked alteration of vascular permeability.

Polymorphonuclear leukocyte infiltration into tissues in host defense and inflammatory disease causes increased vascular permeability and edema formation through unknown mechanisms. Here, we report the involvement of a paracrine mechanism in neutrophil-evoked alteration in endothelial barrier function. We show that upon neutrophil adhesion to the endothelial lining, leukocytic β2 integrin signaling triggers the release of neutrophil-borne heparin-binding protein (HBP), also known as CAP37/azurocidin, a member of the serprocidin family of neutrophil cationic proteins. HBP induced Ca++-dependent cytoskeletal rearrangement and intercellular gap formation in endothelial-cell monolayers in vitro, and increased macromolecular efflux in microvessels in vivo. Moreover, selective inactivation of HBP prevented the neutrophils from inducing endothelial hyperpermeability. Our data suggest a fundamental role of neutrophil-derived HBP in the vascular response to neutrophil trafficking in inflammation. Targeting this molecule in inflammatory disease conditions offers a new strategy for prevention of endothelial barrier dysfunction caused by misdirected leukocyte activation.

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.

Characterization of recombinant human HBP/CAP37/azurocidin, a pleiotropic mediator of inflammation-enhancing LPS-induced cytokine release from monocytes

Neutrophil‐derived heparin‐binding protein (HBP) is a strong chemoattractant for monocytes. We report here for the first time the expression of recombinant HBP. A baculovirus containing the human HBP cDNA mediated in insect cells the secretion of a 7‐residue N‐terminally extended HBP form (pro‐HBP). Deletion of the pro‐peptide‐encoding cDNA sequence resulted in correctly processed HBP at the N‐terminus. Electrospray mass spectrum analysis of recombinant HBP yielded a molecular weight of 27.237 ± 3 amu. Consistent with this mass is a HBP form of 225 amino acids (mature part +3 amino acid C‐terminal extension). The biological activity of recombinant HBP was confirmed by its chemotactic action towards monocytes. Furthermore, we have shown that recombinant HBP stimulates in a dose‐dependent manner the lipopolysaccharide (LPS)‐induced cytokine release from human monocytes.

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 Ca2+ 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.

A neutrophil-derived proteolytic inactive elastase homologue (hHBP) mediates reversible contraction of fibroblasts and endothelial cell monolayers and stimulates monocyte survival and thrombospondin secretion

Human heparin‐binding protein (hHBP) is a recently discovered proteolytically inactive neutrophil elastase homologue with sequence identity to azurocidin and CAP37. The protein has antibacterial properties and chemotactic activity toward monocytes. In the present work, we show that monocytes, cultured under serum‐free conditions, developed morphological changes and formed multicellular aggregates 4 h after the addition of hHBP at a concentration of 10 μg/ml. However, after prolonged incubation (11 days) with unchanged medium, the cells spread again. The hHBP‐treated cells had a two‐ to threefold increase in survival compared to control cells, measured using trypan blue as an indicator of living cells. Differentiation of the alive cells to macrophages was detected by changes in morphology, a threefold increase in protein content, and a three‐ to fourfold increase in acid phosphatase activity. When monocytes in parallel experiments were labelled with [35S]methionine de novo synthesis and secretion of thrombospondin in a dose‐dependent manner was observed after 16 h, with half‐maximal secretion at 2 μg hHBP/ml and a maximal 12‐fold increase in secretion with respect to controls at 16 μg/ml. Supplementary labeling with [35S]sulfate revealed that the same monocytes down‐regulated the secretion of a large proteoglycan (300–400 kd), apparently also with a half‐maximal decrease rate at 2 μg/ml hHBP. Exposure of confluent fibroblast and endothelial cell monolayers to hHPB (10 μg/ml) in the absence of fetal calf serum resulted in cell contraction leaving gaps between cells, the phenomenon being recognizable within 4 h after addition of hHBP. Addition of fetal calf serum to a concentration of 10% completely restored the monolayers. A unique role of hHBP in host defense involving recruitment of monocytes and a key funciton of hHBP in neutrophil extravasation in response to inflammatory chemotactic signals such as leukotriene B4, complement peptide G5a, and N‐formyl‐methionyl‐leucyl‐phenylalanine are suggested.

Diabetic Late Complications: Will Aldose Reductase Inhibitors or Inhibitors of Advanced Glycosylation Endproduct Formation Hold Promise?

Patients suffering from the severe complications associated with both insulin- (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM): nephropathy, retinopathy, neuropathy, and atherosclerosis are still largely left without a prospect of an efficient treatment. This is the case even if it has been assumed for decades and now finally proved by the results from the Diabetes Control and Complications Trial (DCCT) that hyperglycemia is the single main cause of these complications. Improved glycemic control as a result of intensive insulin treatment has the potential to reduce the incidence and progression of complications, but implementation and monitoring of improved glycemic control in all groups of IDDM and NIDDM patients in different communities will be difficult and expensive. Results from the recently terminated DCCT have shown that even with intensive insulin treatment, there will be a significant burden of complications on the diabetic population. It will, therefore, still be of immense importance for the long-term quality of life for the diabetic patient that additional possibilities are developed for prevention and intervention against diabetic complications. Almost two decades of research, animal model testing, and clinical trials have been conducted on various efficient aldose reductase inhibitors. Now the concept of inhibition of formation of advanced glycosylation endproducts on proteins and lipids resulting from extra- and intracellular hyperglycemia is entering the scene as an alternative or perhaps supplementary approach to reduce the occurrence of diabetic complications. An overview of the results from these two fields of research and associated drug-development programs will be presented along with thoughts on possible future developments.

CHARACTERIZATION OF THE BIOSYNTHESIS, PROCESSING, AND SORTING OF HUMAN HBP/CAP37/AZUROCIDIN

Azurocidin is a multifunctional endotoxin‐ binding serine protease homolog synthesized during the promyelocytic stage of neutrophil development. To characterize the biosynthesis and processing of azurocidin, cDNA encoding human preproazurocidin was stably transfected to the rat basophilic leukemia cell line RBL‐1 and the murine myeloblast‐like cell line 32D cl3; cell lines previously utilized to study the related proteins cathepsin G and proteinase 3. After 30 min of pulse radiolabeling, two forms of newly synthesized proazurocidin (34.5 and 37 kDa), differing in carbohydrate content but with protein cores of identical sizes, were recognized. With time, the 34.5‐kDa form disappeared, while the 37‐kDa form was further processed proteolytically, as judged by digestion with N‐glycosidase F. Conversion of high‐mannose oligosaccharides into complex forms was shown by acquisition of complete resistance to endoglycosidase H. Radiosequence analysis demonstrated that the amino‐terminal seven amino acid propeptide of proazurocidin was removed in a stepwise manner during processing; initial removal of five amino acids was followed by cleavage of a dipeptide. Presence of the protease inhibitors Gly‐Phe‐diazomethyl ketone, bestatin, or leupeptin inhibited only the cleavage of the dipeptide, thus indicating the involvement of at least two amino‐ terminal processing enzymes. Translocation of azurocidin to granules was shown by subcellular fractionation. Similar results, with efficient biosynthesis, processing, and targeting to granules in both cell lines, were obtained with a mutant form of human preproazurocidin lacking the amino‐terminal heptapropeptide. In conclusion, this investigation is an important addition to our previous studies on related azurophil granule proteins, and provides novel information concerning the biosynthesis and distinctive amino‐terminal processing of human azurocidin. J. Leukoc. Biol. 66: 634–643; 1999.

HEPARIN BINDING PROTEIN (CAP37) IS AN OPSONIN FOR STAPHYLOCOCCUS AUREUS AND INCREASES PHAGOCYTOSIS IN MONOCYTES

Heparin binding protein (HBP), also known as cationic antibiotic protein (CAP37) or azurocidin, is stored in azurophilic granules of neutrophils and is released to the extracellular space when granulocytes phagocytose Staphylococcus aureus. We investigated whether extracellular HBP also has the potential to increase phagocytosis of S. aureus by other phagocytes. We used flow cytometry to characterize the binding of HBP to S. aureus and to simultaneously measure phagocytosis and superoxide production of opsonized S. aureus in monocytes and granulocytes. Our results demonstrate that HBP is a strong opsonin for S. aureus, and that monocytes, but not granulocytes, increase phagocytosis of HBP-treated S. aureus. However, HBP-treated S. aureus increases the production of superoxide in both monocytes and granulocytes as compared with untreated S. aureus. These findings support the role of granulocytes in the afferent limb of inflammation and demonstrate that HBP, when released from activated granulocytes, potentiates bacterial uptake in monocytes and enhances the potential of microbial killing in monocytes and granulocytes.

Atomic resolution structure of human HBP/CAP37/azurocidin.

Crystals of human heparin binding protein (HBP) diffract to 1.1 A when flash-frozen at 120 K. The atomic resolution structure has been refined anisotropically using SHELXL96. The final model of HBP consists of 221 amino-acid residues of 225 possible, three glycosylation units, one chloride ion, 15 precipitant ethanol molecules and 323 water molecules. The structure is refined to a final crystallographic R factor of 15.9% and Rfree(5%) of 18.9% using all data. A putative protein kinase C activation site has been identified, involving residues 113-120. The structure is compared to the previously determined 2.3 A resolution structure of HBP.

Heparin binding protein increases survival in murine fecal peritonitis.

ObjectiveTo test the effectiveness of recombinant heparin-binding protein (HBP), a neutrophil-derived multifunctional protein with monocytic-specific properties, in fecal peritonitis and polymicrobial sepsis. DesignProspective, controlled animal trial. SettingAnimal research laboratory. SubjectsSwiss Webster mice challenged with cecal ligation and puncture (CLP) and treated with recombinant HBP and 60 mg/kg cefoxitin twice a day. InterventionsHBP was administered to mice at different concentrations and different intervals before and after CLP. Rat albumin (1%) was administered to control animals. Measurements and Main ResultsMortality Rate: Survival was increased in mice pretreated intraperitoneally 24 hrs before CLP with 10 &mgr;g or 100 &mgr;g of HBP without cefoxitin (p = .01, Cox-Mantel log-rank test). Compared with control animals, survival was increased significantly (from 5% to 47%, p = .014) in mice that received cefoxitin and 50 &mgr;g ip HBP immediately after CLP, followed by continuous administration of HBP (12 &mgr;g/24 hrs). Intravenous administration of HBP (0.1, 1, and 10 &mgr;g) at the time of CLP showed an opposite dose effect; low doses (0.1 &mgr;g) prolonged early survival, whereas high dose (10 &mgr;g) shortened survival (p = .036). Compared with control animals, overall survival was not different. Chemotaxis: Cytospin preparations from peritoneal exudate cells (PECs) 48 hrs after administration of 10 &mgr;g and 100 &mgr;g ip HBP demonstrated a 1.7-fold increase in the total number of macrophages compared with carrier control (p < .05). Phagocytosis: A flow cytometric in vitro assay demonstrated that administration of 10 &mgr;g ip HBP alone did not enhance phagocytosis of fluorescent Escherichia coli in PECs. However, 24-hr pretreatment with 10 &mgr;g of HBP followed by CLP increased phagocytosis in PECs 1.8-fold compared with the control CLP group (p = .04). Receptor expression: CD16/CD32w expression in PECs did not change after HBP or CLP. CD11b and CD18 expression in PECs was increased significantly after CLP compared with PECs from non-CLP-challenged animals (p < .05). Pretreatment with 10 &mgr;g of HBP did not further enhance CD11b/CD18 expression in PECs. ConclusionsRecombinant HBP increases survival in murine fecal peritonitis. The mechanisms by which HBP reduces septic death are not fully understood, but they include monocyte chemotaxis and increased phagocytosis of E. coli by PECs. Our data suggest that the inflammatory response induced by CLP is important for the effect of HBP to enhance phagocytosis.