Oliver Wiedow

Elafin is a potent inhibitor of proteinase 3

Elafin, a human skin derived inhibitor of human leukocyte elastase, was tested for inhibitory activity against proteinase 3, an elastin degrading proteinase of neutrophils. The inhibitory activity of elafin was compared with antileukoprotease and eglin C. Elafin proved to be a potent inhibitor of elastin-FITC degradation showing an IC 50 of 9.5 x 10(-9) M. Potency was found to be more than 100-fold higher as compared with antileukoprotease and eglin C.

Attenuated induction of epithelial and leukocyte serine antiproteases elafin and secretory leukocyte protease inhibitor in Crohn’s disease

Elafin (or skin‐derived antileukoprotease) and secretory leukocyte protease inhibitor (SLPI) are serine antiproteases antagonizing human neutrophil elastase (HNE), thereby preventing tissue injury from excessive release of proteolytic enzymes by inflammatory cells. Furthermore, elafin and SLPI are “defensin‐like” molecules with broad antimicrobial activity. The balance between proteases and antagonists may critically determine inflammatory processes in Crohn’s disease (CD) and ulcerative colitis (UC). Real‐time PCR was performed to quantitate colonic, proinflammatory cytokine IL‐8, protease (HNE), and antiprotease mRNA (elafin and SLPI) in a total of 340 biopsies from 117 patients (47 CD, 45 UC, 25 controls). Histological inflammation was scored, and HNE, elafin, and SLPI were localized and semiquantified by immunostaining in 51 colonic paraffin sections (23 CD, 11 UC, 17 controls). Proinflammatory IL‐8, degree of histological inflammation, and granulocyte content were similar in UC and CD. Elafin stained predominantly in the epithelium and SLPI in mucosal inflammatory cells. HNE mRNA levels and immunostaining were increased equally in both forms of inflammatory bowel disease. Levels of mRNA and immunostaining of the antiproteases elafin and SLPI were enhanced strongly in inflamed versus noninflamed UC. It is surprising that comparing inflamed versus noninflamed CD, this increase was significantly less pronounced for elafin and even lacking for SLPI. Despite comparable degrees of inflammation and protease levels, the induction of both antiproteases was attenuated in CD. This could contribute to the transmural depth of tissue destruction in CD. Elafin and SLPI may be added to the list of defensin‐like peptides with diminished induction in CD versus UC.

Neutrophil serine proteases: mediators of innate immune responses.

Purpose of reviewNeutrophil cells have been considered mainly as innate immune cells directed against microbial threats. Their serine proteases neutrophil elastase, proteinase 3 and cathepsin G are main constituents and are released at sites of inflammation. During recent years it became clear that neutrophil serine proteases act as regulators of cell signaling and immune regulation. Recent findingsNeutrophils are able to form so-called neutrophil extracellular traps. Recent studies showed that these extracellular traps might be involved in small vessel vasculitis and lupus nephritis. Neutrophil serine proteases in concert with externalized nucleosomes promote thrombus formation inside blood vessels. This event helps retain bacteria inside liver microvessels and thereby prevents the extravasation of pathogens. Moreover, neutrophil serine proteases act as alternative processing enzymes of pro-inflammatory cytokines IL-1β and IL-18 in vivo and modulate other inflammation-related control mechanisms such as progranulin inactivation, matrix metalloproteinase-9 activation and IL-6 inactivation. Recent studies point to an involvement of neutrophil elastase in lung cancer by inducing mitogenesis after entering the cells. SummaryThe knowledge of the different functions of neutrophils is still expanding. Recent findings underline the importance of neutrophil serine proteases as key mediators of inflammatory processes and point to novel strategies against inflammatory disorders.

Supernatants of Pseudomonas aeruginosa induce the Pseudomonas-specific antibiotic elafin in human keratinocytes

Abstract: Elafin is a skin‐derived serine‐protease inhibitor. It is thought to be important to prevent human leukocyte elastase‐mediated tissue damage and might play an important role in maintaining the integrity of the human epidermis. Recent studies have provided evidence for an antimicrobial activity of elafin against P. aeruginosa. As gram‐negative infections typically occur in barrier‐disrupted skin we were interested to determine whether supernatants of the gram‐negative bacteria P. aeruginosa and Escherichia coli were capable of inducing elafin expression.

Therapeutic potential of human elafin

Elafin is an endogenous human protein composed of an N-terminal transglutaminase substrate motif and a C-terminal WAP (whey acidic protein)-domain with antiproteolytic properties. Elafin is expressed predominantly in epithelial tissue and potently inhibits the neutrophil-derived serine proteases elastase and proteinase-3 by a competitive tight-binding mechanism. Furthermore, it inhibits EVE (endogenous vascular elastase). Studies on several animal models show that antiprotease augmentation with human elafin is an effective strategy in the treatment of inflammatory vascular, systemic and pulmonary diseases and of inflammation triggered by reperfusion injury. This raises the possibility that elafin might be effective in the treatment of a variety of human inflammatory diseases. In a Phase I clinical trial, elafin was well tolerated. Phase II trials are underway to investigate the therapeutic effects of elafin on post-operative inflammation and the clinical consequences of major surgery. Of particular interest is the reduction of post-operative morbidity after oesophagus cancer surgery, coronary artery bypass surgery and kidney transplantation.

Elafin is Specifically inactivated by RgpB from Porphyromonas gingivalis by Distinct Proteolytic Cleavage

Abstract Porphyromonas gingivalis, the major causative bacterium of periodontitis, contributes significantly to elevated proteolytic activity at periodontal pockets owing to the presence of both bacteria and host, predominantly neutrophil-derived, serine proteases. Normally the activity of the latter enzymes is tightly regulated by endogenous proteins, including elafin, a potent neutrophil elastase and proteinase 3 inhibitor released from epithelial cells at sites of inflammation. Here, we report that all three gingipains (HRgpA, RgpB, and Kgp) have the ability to degrade elafin, with RgpB being far more efficient than other gingipains. RgpB efficiently inactivates the inhibitory activity of elafin at subnanomolar concentrations through proteolysis limited to the Arg22-Cys23 peptide bond within the surface loop harboring the inhibitor active site. Notably, elafin resists inactivation by several Staphylococcus aureus-derived serine and cysteine proteases, confirming the high stability of this protein against proteolytic degradation. Therefore, we conclude that elafin inactivation by RgpB represents a specific pathogenic adaptation of P. gingivalis to disturb the protease-protease inhibitor balance in the infected gingival tissue. This contributes to enhanced degradation of host proteins and generation of a pool of peptides serving as nutrients for this asaccharolytic pathogen.

Lesional elastase activity in psoriasis

Human leukocyte elastase, a neutrophil-derived serine protease, is present in psoriatic lesions in an enzymatically active form. Our purpose was to assess the significance of human leukocyte elastase determinations in estimating the inflammatory activity of psoriatic lesions. A standardized method was used to analyse lesional elastase activity. Elastase activities were correlated with erythema, induration and hyperkeratosis of psoriatic lesions in 54 patients. Lesional elastase activities were also determined during treatment with salt-water bathing and UVB irradiation. Lesional elastase activity correlated with skin induration and was inversely correlated with hyperkeratosis of the lesions. Psoriatic lesions with high elastase activity responded well to therapy, whereas lesions with low elastase activity appeared to be comparatively resistant. This study shows that by quantitative determination of lesional elastase activities it is possible to distinguish predominantly inflammatory from predominantly hyperproliferative psoriasis. The latter shows delayed responsiveness to topical therapy with salt-water bathing plus UVB irradiation.

Human leukocyte elastase and cathepsin G are specific inhibitors of C5a-dependent neutrophil enzyme release and chemotaxis.

Abstract:  Circulating human neutrophils from patients with severe inflammatory disorders such as erysipelas and sepsis are specifically desensitized to complement factor C5a stimulation but not to stimulation with other stimuli like N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP), interleukin‐8 (IL‐8), leukotriene B4 (LTB4), or platelet‐activating factor (PAF, 1‐O‐alkyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine). In this study, we raised the question whether factors released from polymorphonuclear leukocytes (PMNs) can specifically down‐regulate C5a‐dependent neutrophil functions.

Psoriasis scales contain C5a as the predominant chemotaxin for monocyte-derived dendritic cells.

Abstract: Dendritic cells seem to be of major importance for the pathogenesis of psoriasis. They are increased in number in lesional psoriatic skin which is thought to be due to an increased influx from the peripheral blood regulated by chemotaxins. Using a biological/biochemical approach we have addressed the question whether psoriasis scale extracts contain proteinaceous chemotaxins for dendritic cells. Human monocytes differentiated into dendritic cells by culture with GM‐CSF and IL‐4 (MoDC) served as responder cells. Chemotactic activity for MoDC was purified by several HPLC‐steps. The results of our study show that C5a/C5adesarg is the major chemotactic peptide for MoDC in psoriasis scale extracts. In comparison to other stimuli such as fMLP or monocyte chemotactic peptide 1 (MCP‐1) C5a proved to be a most potent and efficient chemotaxin for MoDC. C5a co‐eluted with MRP14/calgranulin B which is present in large amounts in psoriasis scale extracts as identified by amino acid sequencing. However, MRP14/calgranulin B did not possess any chemotactic activity for MoDC. Our results provide evidence that C5a/C5adesarg although not specific for dendritic cells seems to be the major chemoattractant for these cells in lesional psoriasis skin.

Trypsin induces epidermal proliferation and inflammation in murine skin

Abstract:  Human keratinocytes are known to express the protease‐activated receptors, PAR‐1 and PAR‐2. Activation of PAR‐1 results in increased proliferation, whereas PAR‐2 activation results in decreased keratinocyte proliferation. Trypsin activates PAR‐1 and in higher concentrations, PAR‐2. The aim of this study was to evaluate the overall effect of trypsin on keratinocyte proliferation in a mouse in vivo and in vitro model. Daily topical application of 0.3–300 pmol trypsin/cm2 on hairless mouse skin induced dose‐dependent epidermal hyperproliferation as determined by an increase in 5‐bromo‐2′‐deoxyuridine incorporation of up to eight‐fold in basal keratinocytes and an up to three‐fold increase in keratinocyte layers. This was accompanied by an increased transepidermal water loss. These effects of trypsin were abolished by the addition of the trypsin inhibitor n‐p‐tosyl‐l‐lysine‐chloromethyl ketone. Histological analysis revealed acanthosis, hypergranulosis, and spongiosis in the epidermis as well as vasodilatation and an inflammatory infiltrate in the upper dermis. In the murine keratinocyte cell line PAM‐212 activation of PAR‐1 with specific activating peptides resulted in a calcium influx and an increase of proliferation, whereas activation of PAR‐2 caused a diminished proliferation. Incubation with trypsin, PAR‐1‐, and PAR‐2‐activating peptides induced cytokine‐induced neutrophil chemoattractant (KC) mRNA expression as a marker for inflammation in PAM‐212 in a dose‐dependent manner. In conclusion, our results suggest that trypsin induces in vivo epidermal proliferation and inflammation. Proliferation seems not to be signaled by PAR activation, but PAR‐2‐induced KC chemokine expression may contribute in part to trypsin‐induced inflammation.