The role of neutrophil granule proteins in neuroinflammation and Alzheimer's disease.

Neutrophils are the innate immune system's first line of defense. Neutrophils play a critical role in protecting the host against infectious pathogens, resolving sterile injuries, and mediating inflammatory responses. The granules of neutrophils and their constituent proteins are central to these functions. Although neutrophils may exert a protective role upon acute inflammatory conditions or insults, continued activity of neutrophils in chronic inflammatory diseases can contribute to tissue damage. Neutrophil granule proteins are involved in a number of chronic inflammatory conditions and diseases. However, the functions of these proteins in neuroinflammation and chronic neuroinflammatory diseases, including Alzheimer's disease (AD), remain to be elucidated. In this review, we discuss recent findings from our lab and others that suggest possible functions for neutrophils and the neutrophil granule proteins, CAP37, neutrophil elastase, and cathepsin G, in neuroinflammation, with an emphasis on AD. These findings reveal that neutrophil granule proteins may exert both neuroprotective and neurotoxic effects. Further research should determine whether neutrophil granule proteins are valid targets for therapeutic interventions in chronic neuroinflammatory diseases.

Differences in galectin-3, a biomarker of fibrosis, between participants with peripheral artery disease and participants with normal ankle-brachial index.

The aim of this study was to determine if galectin-3 levels were different between participants with peripheral artery disease (PAD) and controls, and to describe its relationship with markers of early atherosclerosis. Sixty participants were recruited into two groups: a PAD group (n=31), ankle-brachial index (ABI) ⩽0.90 and a normal ABI group (n=29), ABI 1.0-1.4. PAD participants were older (68.6 vs 61.8 years, p=0.037), more commonly men (68% vs 38%, p=0.02), and with more cardiovascular risk factors (p<0.001). Galectin-3 was 22% higher in PAD participants (mean±SD: 17.6±4.7 vs 14.4±4.1 ng/mL, p<0.01). The odds ratio for galectin-3 in PAD to be 1 ng/mL higher than the participants with normal ABI was 1.19, after adjusting by age and gender (p=0.014). High-sensitivity C-reactive protein (hs-CRP) and homeostatic model assessment (HOMA) were positively associated with galectin-3 in the age- and gender-adjusted model, while arterial elasticity and microalbuminuria were not. In conclusion, galectin-3 levels were higher in participants with PAD.

Selecting Multitarget Peptides for Alzheimer's Disease.

Alzheimer's disease (AD) is a multifactorial disease with a complex pathogenesis. Developing multitarget drugs could be a powerful strategy to impact the progressive loss of cognitive functions in this disease. The purpose of this study is to select a multitarget lead peptide candidate among a series of peptide variants derived from the neutrophil granule protein cathepsin G. We screened eight peptide candidates using the following criteria: (1) Inhibition and reversion of amyloid beta (Aß) oligomers, quantified using an enzyme-linked immunosorbent assay (ELISA); (2) direct binding of peptide candidates to the human receptor for advanced glycation end-products (RAGE), the Toll-like receptor 4 (TLR4) and the S100 calcium-binding protein A9 (S100A9), quantified by ELISA; (3) protection against Aß oligomer-induced neuronal cell death, using trypan blue to measure cell death in a murine neuronal cell line; (4) inhibition of TLR4 activation by S100A9, using a human TLR4 reporter cell line. We selected a 27-mer lead peptide that fulfilled these four criteria. This lead peptide is a privileged structure that displays inherent multitarget activity. This peptide is expected to significantly impact cognitive decline in mouse models of Alzheimer's disease, by targeting both neuroinflammation and neurodegeneration.

Candidacidal activity of synthetic peptides based on the antimicrobial domain of the neutrophil-derived protein, CAP37.

The primary bactericidal domain of CAP37, a cationic antimicrobial protein with potent activity against Gram-negative organisms was previously shown to reside between amino acids 20 through 44 (NQGRHFCGGALIHARFVMTAASCFQ) of the native protein. In this study, we explored the efficacy of four synthetic CAP37 peptide analogs, based on this sequence, against various Candida species including fluconazole-sensitive and -resistant isolates of C. albicans. Three of the peptides demonstrated strong antifungal activity for C. albicans, including fluconazole-resistant isolates of C. albicans and were active against C. guilliermondii, C. tropicalis, C. pseudotropicalis, C. parapsilosis, and C. dubliniensis. The peptides were ineffective against C. glabrata, C. krusei, and Saccharomyces cerevisiae. For C. albicans isolates, the peptides had relatively greater activity against blastoconidia than hyphal forms, although strong antifungal activity was observed with pseudohyphal forms of the various Candida species tested. Kinetic studies demonstrated fungicidal rather than fungistatic activity. These findings indicate that synthetic peptides based on the antimicrobial domain of CAP37 also have activity against eukaryotic organisms suggesting a broader range of activity than originally demonstrated and show for the first time their potent fungicidal activity.

Antimicrobial Peptides Derived from the Immune Defense Protein CAP37 Inhibit TLR4 Activation by S100A9.

Purpose: Corneal abrasion is a common eye injury, and its resolution can be seriously complicated by bacterial infection. We showed that topical application of the cationic antimicrobial protein of 37 kDa (CAP37) promotes corneal re-epithelialization in mice, and peptides derived from CAP37 can recapitulate the antibacterial and wound-healing effects of the full-length protein. The current study was designed to identify the molecular mechanisms mediating the wound-healing effect of CAP37 and derived bioactive peptides. Methods: We used a TriCEPS-based, ligand-receptor glycocapture method to identify the binding partners of CAP37 on live human corneal epithelial cells using the hTCEpi cell line. We used an ELISA method to confirm binding with identified partners and test the binding with CAP37-derived peptides. We used a reporter cell line to measure activation of the identified membrane receptor by CAP37 and derived peptides. Results: We pulled down S100 calcium-binding protein A9 (S100A9) as a binding partner of CAP37 and found that CAP37 and four derived peptides encompassing two regions of CAP37 bind S100A9 with high affinities. We found that CAP37 and the S100A9-binding peptides could also directly interact with the Toll-like receptor 4 (TLR4), a known receptor for S100A9. CAP37 and one peptide partially activated TLR4. The other three peptides did not activate TLR4. Finally, we found that CAP37 and all four peptides could inhibit the activation of TLR4 by S100A9. Conclusions: This study identifies a mechanism of action for CAP37 and derived antimicrobial peptides that may restrain inflammatory responses to corneal injury and favor corneal re-epithelialization.

Bioactive Antimicrobial Peptides as Therapeutics for Corneal Wounds and Infections.

Significance: More than 2 million eye injuries and infections occur each year in the United States that leave civilians and military members with reduced or complete vision loss due to the lack of effective therapeutics. Severe ocular injuries and infections occur in varied settings including the home, workplace, and battlefields. In this review, we discuss the potential of developing antimicrobial peptides (AMPs) as therapeutics for the treatment of corneal wounds and infections for which the current treatment options are inadequate. Recent Advances: Standard-of-care employs the use of fluorescein dye for the diagnosis of ocular defects and is followed by the use of antibiotics and/or steroids to treat the infection and reduce inflammation. Recent advances for treating corneal wounds include the development of amniotic membrane therapies, wound chambers, and drug-loaded hydrogels. In this review, we will discuss an innovative approach using AMPs with the dual effect of promoting corneal wound healing and clearing infections. Critical Issues: An important aspect of treating ocular injuries is that treatments need to be effective and administered expeditiously. This is especially important for injuries that occur during combat and in individuals who demonstrate delayed wound healing. To overcome gaps in current treatment modalities, bioactive peptides based on naturally occurring cationic antimicrobial proteins are being investigated as new therapeutics. Future Directions: The development of new therapeutics that can treat ocular infections and promote corneal wound healing, including the healing of persistent corneal epithelial defects, would be of great clinical benefit.

Effect of Cationic Antimicrobial Protein CAP37 on Cytokine Profile during Corneal Wound Healing.

The cationic antimicrobial protein of 37 kDa (CAP37) mediates proliferation, migration, and adhesion of human corneal epithelial cells and promotes corneal re-epithelialization in mouse. The purpose of this study was to investigate the cytokine profile following abrasion of the corneal epithelium, and to identify the cytokines modulated by topical treatment with CAP37 to determine the mechanism by which CAP37 contributes to the recruitment of inflammatory cells and healing of the cornea. The corneal epithelium in mouse eyes was removed and wounds were treated with a saline vehicle or human recombinant CAP37. Wounds were visualized with fluoresce in staining at 0, 16, 24 and 48 h. Mouse corneas were excised at 0, 6, 16, 24 and 48 h post corneal abrasion. The excised corneas were analyzed by immunohistochemistry for re-epithelialization and infiltration of inflammatory cells while the expression profiles of thirty-two cytokines were investigated by multiplex analysis. Results corroborating previous studies showed accelerated wound closure in corneas treated with CAP37 compared to those treated with the saline vehicle. Immunohistochemistry revealed less neutrophil infiltration in CAP37-treated corneas when compared to controls at 24 h. By 48 h post-wounding, histological analysis revealed more staining for neutrophils than the staining observed in the controls. Modulation of cytokine expression occurred for the majority of the cytokines tested at the time of corneal abrasion, during re-epithelialization, and/or by CAP37 treatment. Cytokines monocyte chemoattractant protein-1 (MCP-1) and regulated on activation, normal T cell expressed and secreted (RANTES) were induced during re-epithelialization, at the early 16 h time point. Interleukin 6 (IL-6), leukemia inhibitory factor (LIF), granulocyte colony-stimulating factor (G-CSF), IL-12p70, macrophage inflammatory protein 1 beta (MIP-1ß), and interferon gamma-induced protein 10 (IP-10) were induced at 24 h and unchanged during CAP37 treatment. By contrast, IL-15, monokine induced by gamma interferon (MIG), keratinocyte-derived cytokine (KC), tumor necrosis factor alpha (TNF-α), MIP-1α, IL-1ß, and macrophage colony-stimulating factor (M-CSF) were modulated by CAP37 treatment. In general, CAP37 appeared to decrease pro-inflammatory cytokines at 24 h and increase them at 48 h when compared to the control group. These data demonstrate that CAP37 modulates the production of cytokines in the cornea and suggest that limiting the number of neutrophils recruited during the early inflammatory phase may support corneal re-epithelialization.

Novel therapies based on cationic antimicrobial peptides.

Cationic antimicrobial peptides serve as critical defense molecules protecting the host from invading bacteria, viruses and fungi. These antimicrobial peptides are widely distributed in nature and in vertebrates they have been localized to numerous tissues and cells. Cationic antimicrobial peptides can be expressed constitutively or under certain circumstances they can be induced in response to infection, inflammatory mediators, and cytokines. Although, their original and primary function was believed to be antimicrobial, it is now becoming clear that these antimicrobial peptides have a wide repertoire of functions with interesting ramifications on the immune system that are not solely antimicrobial. An area of active research is the determination of the mechanism(s) of action of antimicrobial peptides which have yet to be clarified. However, current consensus is that the mechanism is sufficiently different from conventional antibiotics that the development of resistance could be remote. Their broad spectrum activity, low potential to induce resistance and diverse functions make antimicrobial peptides an attractive family of compounds that have potential to be developed as therapeutics for treating certain infections.

The Role of Neutrophil Proteins on the Amyloid Beta-RAGE Axis.

We previously showed an elevated expression of the neutrophil protein, cationic antimicrobial protein of 37kDa (CAP37), in brains of patients with Alzheimer's disease (AD), suggesting that CAP37 could be involved in AD pathogenesis. The first step in determining how CAP37 might contribute to AD pathogenesis was to identify the receptor through which it induces cell responses. To identify a putative receptor, we performed GAMMA analysis to determine genes that positively correlated with CAP37 in terms of expression. Positive correlations with ligands for the receptor for advanced glycation end products (RAGE) were observed. Additionally, CAP37 expression positively correlated with two other neutrophil proteins, neutrophil elastase and cathepsin G. Enzyme-linked immunosorbent assays (ELISAs) demonstrated an interaction between CAP37, neutrophil elastase, and cathepsin G with RAGE. Amyloid beta 1-42 (Aß1-42), a known RAGE ligand, accumulates in AD brains and interacts with RAGE, contributing to Aß1-42 neurotoxicity. We questioned whether the binding of CAP37, neutrophil elastase and/or cathepsin G to RAGE could interfere with Aß1-42 binding to RAGE. Using ELISAs, we determined that CAP37 and neutrophil elastase inhibited binding of Aß1-42 to RAGE, and this effect was reversed by protease inhibitors in the case of neutrophil elastase. Since neutrophil elastase and cathepsin G have enzymatic activity, mass spectrometry was performed to determine the proteolytic activity of all three neutrophil proteins on Aß1-42. All three neutrophil proteins bound to Aß1-42 with different affinities and cleaved Aß1-42 with different kinetics and substrate specificities. We posit that these neutrophil proteins could modulate neurotoxicity in AD by cleaving Aß1-42 and influencing the Aß1-42 -RAGE interaction. Further studies will be required to determine the biological significance of these effects and their relevance in neurodegenerative diseases such as AD. Our findings identify a novel area of study that underscores the importance of neutrophils and neutrophil proteins in neuroinflammatory diseases such as AD.

Is the Use of Complete Blood Counts with Manual Differentials an Antiquated Method of Determining Neutrophil Composition in Newborns?

BACKGROUND: Automated complete blood counts (CBCs) provide imprecise measures of neonatal neutrophil composition due to the presence of nucleated red cells, which masquerade as white cells. Manual differentials (MDs) must be performed but can be flawed due to technician inexperience or limited cell counts. Our study evaluated whether flow cytometry could more accurately determine neutrophil composition in term newborns compared with adult controls. METHODS: Neutrophils were obtained by venipuncture in adults (n=10) and via cord blood from neonates delivered vaginally (n=21) or by primary cesarean section (n=17). Samples were processed by both flow cytometry and CBC with MD and results compared. Flow cytometry findings were verified using gene expression analysis of granule proteins. RESULTS: Immature-to-total neutrophil ratios were used to signify neutrophil composition. Flow cytometry was superior to manual differentials due to its ability to identify the earliest neutrophil forms. Significant differences between newborns and adults were determined using flow cytometry (0.12 versus 0.05; p=0.01), but not MDs (p=0.18). Neonates had similar findings irrespective of labor and method used. Verification of neutrophil maturity was completed using gene expression analysis of granule proteins. CONCLUSIONS: These results raise concerns about the accuracy of using CBCs with MDs to determine neutrophil composition. Further investigation of flow cytometry in clinical practice is warranted.

A multifunctional peptide based on the neutrophil immune defense molecule, CAP37, has antibacterial and wound-healing properties.

CAP37, a protein constitutively expressed in human neutrophils and induced in response to infection in corneal epithelial cells, plays a significant role in host defense against infection. Initially identified through its potent bactericidal activity for Gram-negative bacteria, it is now known that CAP37 regulates numerous host cell functions, including corneal epithelial cell chemotaxis. Our long-term goal is to delineate the domains of CAP37 that define these functions and synthesize bioactive peptides for therapeutic use. We report the novel finding of a multifunctional domain between aa 120 and 146. Peptide analogs 120-146 QR, 120-146 QH, 120-146 WR, and 120-146 WH were synthesized and screened for induction of corneal epithelial cell migration by use of the modified Boyden chamber assay, antibacterial activity, and LPS-binding activity. In vivo activity was demonstrated by use of mouse models of sterile and infected corneal wounds. The identity of the amino acid at position 132 (H vs. R) was important for cell migration and in vivo corneal wound healing. All analogs demonstrated antimicrobial activity. However, analogs containing a W at position 131 showed significantly greater antibacterial activity against the Gram-negative pathogen Pseudomonas aeruginosa. All analogs bound P. aeruginosa LPS. Topical administration of analog 120-146 WH, in addition to accelerating corneal wound healing, effectively cleared a corneal infection as a result of P. aeruginosa. In conclusion, we have identified a multifunctional bioactive peptide, based on CAP37, that induces cell migration, possesses antibacterial and LPS-binding activity, and is effective at healing infected and noninfected corneal wounds in vivo.

Modulation of corneal epithelial cell functions by the neutrophil-derived inflammatory mediator CAP37.

PURPOSE: To investigate the effect of CAP37, an inflammatory mediator in neutrophils, on three important events in corneal wound healing: proliferation, migration, and adhesion. METHODS: Immortalized human corneal epithelial cells (HCEC) were treated with CAP37, and its effects on migration and proliferation were measured using the modified Boyden chemotaxis chamber and the proliferation assays (CyQUANT; Molecular Probes, Eugene, OR), respectively. Effects on adhesion were determined by measuring upregulation of adhesion molecules belonging to the selectin, integrin, and immunoglobulin superfamily using RT-PCR and flow cytometry. RESULTS: CAP37 promoted proliferation of HCEC in a time- and dose-dependent fashion. CAP37 was maximally chemotactic for HCEC over a range of 1.3 x 10(-8) to 5.2 x 10(-8) M. CAP37 upregulated intercellular adhesion molecule (ICAM)-1, platelet endothelial cell adhesion molecule (PECAM)-1, and integrin molecules alpha3 (CD49c) and beta1 (CD29). Data on migration and ICAM-1 and PECAM-1 upregulation were corroborated using primary human corneal epithelial cells. CONCLUSIONS: CAP37 modulated corneal epithelial cell proliferation and migration and upregulated adhesion molecules involved in leukocyte-epithelial and epithelial-extracellular matrix interactions.

Corneal expression of the inflammatory mediator CAP37.

PURPOSE: CAP37 is a polymorphonuclear neutrophil (PMN)-derived inflammatory protein with potent antibiotic and chemotactic activity. To further investigate the biological significance of CAP37 in infection and inflammation, a well-characterized in vivo rabbit model of bacterial keratitis was selected to study its contribution to host defenses. METHODS: One hundred colony-forming units of log phase Staphylococcus aureus was injected intrastromally. Eyes were enucleated at 5 to 25 hours after infection and CAP37 detected by immunohistochemistry. To identify the mechanism of CAP37 upregulation in corneal epithelium, in vitro studies using immortalized human corneal epithelial cells (HCECs) were undertaken to determine whether proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), induce CAP37. Because adhesion of leukocytes is important in leukocyte-epithelium interactions, the effect of CAP37 on expression of intercellular adhesion molecule (ICAM)-1 on HCECs was determined by flow cytometry. RESULTS: Strong staining for CAP37 was demonstrated in the corneal epithelium, stromal fibroblasts, ciliary epithelium, related limbus, ciliary vascular endothelium, and bulbar conjunctiva in rabbits injected with S. aureus. The most dramatic expression of CAP37 aside from that in the PMNs occurred in the corneal epithelium. The in vitro studies suggest that CAP37 induction is regulated by TNF-alpha and IL-1beta. In addition, ICAM-1 expression on HCECs was increased in response to CAP37. Molecular cloning of corneal epithelial CAP37 indicated strong sequence identity with an extensive region of PMN-CAP37. CONCLUSIONS: The findings in this study describe the extraneutrophilic expression of CAP37 in response to infection and suggest a role for CAP37 in host defense against infection in the eye.

Corneal wound healing, a newly identified function of CAP37, is mediated by protein kinase C delta (PKCδ).

PURPOSE: The neutrophil-derived granular protein, CAP37, an innate immune system molecule, has antibiotic and immunomodulatory effects on host cells, including corneal epithelial cells. We previously showed that CAP37 modulates corneal epithelial cell migration, adhesion, and proliferation, and that protein kinase C delta (PKCδ) mediates CAP37-induced chemotaxis of these cells. The objective of this study was to investigate the hypothesis that CAP37 facilitates corneal wound healing through the PKC signaling pathway. METHODS: The standard "scratch" assay performed on monolayers of corneal epithelial cells was used to measure the in vitro effect of CAP37 on wound closure. In vivo wound healing in response to CAP37 was measured using a mouse corneal epithelium abrasion model. In vitro and in vivo wound closure were monitored over 48 hours. The PKCδ was visualized during wound closure in cell monolayers and corneal epithelium by immunohistochemistry. The importance of PKCδ in CAP37-induced corneal wound healing was assessed by siRNA. RESULTS: We found that CAP37 accelerated wound closure in vitro and in vivo. Maximal closure occurred with concentrations of CAP37 between 250 and 500 ng/mL. Topical applications on mouse cornea led to re-epithelialization of the cornea by 24 hours. Immunohistochemistry of in vitro and in vivo wounds revealed a local increase of PKCδ along the wound edge in CAP37-treated cell monolayers and corneas, compared to untreated controls. CAP37-induced corneal wound healing was significantly reduced in vivo upon treatment with PKCδ siRNA. CONCLUSIONS: These findings support the hypothesis that CAP37 facilitates corneal wound healing through the activation of PKCδ.

CAP37 activation of PKC promotes human corneal epithelial cell chemotaxis.

PURPOSE: The objective of this study was to elucidate the signaling pathway through which cationic antimicrobial protein of 37 kDa (CAP37) mediates human corneal epithelial cell (HCEC) chemotaxis. METHODS: Immortalized HCECs were treated with pertussis toxin (10 and 1000 ng/mL), protein kinase C (PKC) inhibitors (calphostin c, 50 nM and Ro-31-8220, 100 nM), phorbol esters (phorbol 12,13-dibutyrate, 200 nM and phorbol 12-myristate 13-acetate, 1 µM) known to deplete PKC isoforms, and siRNAs (400 nM) before a modified Boyden chamber assay was used to determine the effect of these inhibitors and siRNAs on CAP37-directed HCEC migration. PKCδ protein levels, PKCδ-Thr(505) phosphorylation, and PKCδ kinase activity was assessed in CAP37-treated HCECs using immunohistochemistry, Western blotting, and a kinase activity assay, respectively. RESULTS: Chemotaxis studies revealed that treatment with pertussis toxin, PKC inhibitors, phorbol esters, and siRNAs significantly inhibited CAP37-mediated chemotaxis compared with untreated controls. CAP37 treatment increased PKCδ protein levels and led to PKCδ phosphorylation on residue Thr(505). Direct activation of PKCδ by CAP37 was demonstrated using a kinase activity assay. CONCLUSIONS: These findings lead us to conclude that CAP37 is an important regulator of corneal epithelial cell migration and mediates its effects through PKCδ.

CAP37-derived antimicrobial peptides have in vitro antiviral activity against adenovirus and herpes simplex virus type 1.

PURPOSE: The antiviral activity of an established antibacterial CAP37 domain and its extracellular mechanism of action were investigated. METHODS: CAP37-derived peptides modified to assess the importance of disulfide bonds were evaluated in cytotoxicity and antiviral assays (direct time kill, dose dependency, and TOTO-1) for adenovirus (Ad) and herpes simplex virus type 1 (HSV-1). RESULTS: Variable virus, adenovirus serotype-dependent, and dose-dependent inhibition were demonstrated without cytotoxicity. For peptide A (CAP37(20-44)), TOTO-1 dye uptake was demonstrated for Ad5 and HSV-1. CONCLUSIONS: Unlike the antibacterial activity of this CAP37 domain, its antiviral activity is not fully dependent upon disulfide bond formation. Viral inhibition appears to result, in part, from disruption of the envelope and/or capsid.

Activation of microglia: a neuroinflammatory role for CAP37.

Recent evidence suggests that inflammation and immune function in the central nervous system (CNS) may play a considerable role in the progression of many neurodegenerative diseases. It is known that microglia, the CNS equivalent of peripheral blood monocytes, may be instrumental in causing neurotoxicity. However, the mediator(s) that activates microglia to produce toxic substances that orchestrate cell death has yet to be elucidated. We have identified a novel inflammatory molecule, cationic antimicrobial protein of molecular weight 37 kDa (CAP37), to the brains of patients dying from Alzheimer's disease. CAP37 is known to be a potent activator and regulator of monocyte function in the systemic circulation. We hypothesize that CAP37, a mediator previously shown to recruit and activate monocytes in the systemic circulation, may also play a role in CNS inflammation by modulating microglial function. Here we demonstrate that CAP37 is a chemoattractant for microglia and that CAP37-treated microglia express class II major histocompatibility antigens and produce proinflammatory cytokines and chemokines. We conclude that CAP37 has the ability to activate microglial cells and suggest that it has the potential to serve as a neuroinflammatory molecule.

CAP37, a neutrophil-derived inflammatory mediator, augments leukocyte adhesion to endothelial monolayers.

Cationic antimicrobial protein of molecular weight 37 kDa (CAP37) is a multifunctional inflammatory mediator that was originally isolated from human neutrophils and described to possess bactericidal and monocyte-activating functions. More recently its expression in endothelial and epithelial cells in response to inflammatory mediators and its ability to activate endothelial cells and alter permeability has been demonstrated. We hypothesize that CAP37 facilitates the process of transendothelial migration not only because of its potential to act as a chemoattractant but also through its ability to promote leukocyte adhesion to the endothelium by modulating adhesion molecule expression on the endothelium. Here we describe its ability to mediate neutrophil and monocyte adherence to endothelial monolayers in vitro. Using reverse transcriptase-polymerase chain reaction and flow cytometry, we demonstrate its ability to upregulate the adhesion molecules, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin in human umbilical vein and lung microvessel endothelial cells. The identity and kinetics of upregulation of the specific adhesion molecule was dependent on the endothelial cell type, suggesting that adhesion molecules on endothelial cells from different vascular beds are differentially regulated by CAP37. The cell-specific kinetics of adhesion molecule upregulation by CAP37 may influence selective leukocyte migration in certain inflammatory situations.

Functional modulation of smooth muscle cells by the inflammatory mediator CAP37.

CAP37, a neutrophil-derived protein, originally identified for its antimicrobial activity is now known to have strong immunoregulatory effects on host cells. Recently, we described its expression and localization within the vascular endothelium associated with atherosclerotic plaques. Since CAP37 is a potent activator of endothelial cells and monocytes, two of the key cellular components of the atherosclerotic plaque, this study was undertaken to determine whether CAP37 had functional effects on smooth muscle cells another important cellular participant in atherosclerosis. Sections from atherosclerotic lesions were stained for the presence of CAP37 and smooth muscle cell alpha actin. The effect of CAP37 on aorta smooth muscle cell migration and proliferation was investigated and the upregulation of adhesion molecules was determined. Immunocytochemistry indicated that CAP37 was present in a subset of smooth muscle cells within atherosclerotic lesions, but was absent in normal vessels. Flow cytometry using double labeling for the proliferation marker Ki-67 and CAP37 demonstrates that CAP37 is mainly expressed in proliferating smooth muscle cells. We show that CAP37 supports migration and proliferation of smooth muscle cells in vitro. Furthermore, CAP37-treated smooth muscle cells expressed higher levels of the cell adhesion molecule ICAM-1 when compared with untreated cells. We suggest that due to its localization to atherosclerotic plaques and its ability to modulate smooth muscle cells, CAP37 may play a role in the progression of this disease.

CAP37, a novel inflammatory mediator: its expression in endothelial cells and localization to atherosclerotic lesions.

Cationic antimicrobial protein of 37 kd (CAP37), originally isolated from human neutrophils, is an important multifunctional inflammatory mediator. Here we describe its localization within the vascular endothelium associated with atherosclerotic plaques. Evidence from in vitro immunocytochemical, Northern blot, and reverse transcriptase-polymerase chain reaction analysis indicates that CAP37 is induced in endothelial cells in response to inflammatory mediators. Endothelial-derived CAP37 shows sequence identity with an extensive region of neutrophil-derived CAP37. This is the first demonstration of endogenous endothelial CAP37, confirmed by sequence analysis. We suggest that, because of its induction and location in the endothelium and its known monocyte- and endothelial-activating capabilities, CAP37 has potential to modulate monocyte/endothelial dynamics at the vessel wall in inflammation.