Identification of elastase in human eosinophils: immunolocalization, isolation, and partial characterization.

Although an elastolytic activity in eosinophil-rich cell fractions from mice has been reported, this enzyme has not been purified and characterized as yet in any mammalian species. Eosinophilic elastase was isolated from human eosinophil fragments (cytosomes) obtained from normal and eosinophilic subjects. The enzyme was purified to apparent electrophoretic homogeneity by fast protein liquid chromatography. The enzyme shows the same physical properties of the major elastase isoenzyme of human neutrophils. In addition, like monocyte elastase, it reacts with a monoclonal antibody against human neutrophil elastase. The biochemical similarities observed between the above-mentioned enzymes and the immunolocalization findings strongly support the idea that human eosinophils and neutrophils contain the same enzyme activity. Eosinophils show immunoreactive material in both types of dense cytoplasmic granules. This observation supports the current hypothesis that the different types of eosinophilic granules represent successive morphological stages of maturation.     

Monoclonal antibodies specific for neutrophil proteinase 4. Production and use for isolation of the enzyme

Four stable hybridoma cell lines producing monoclonal antibodies specific for neutrophil proteinase 4 (NP4) were established and one monoclonal antibody was chosen to produce an immunoaffinity-resin for the purification of NP4. In a precipitation assay system these antibodies bound NP4 in a dose-dependent manner, but did so neither with neutrophil elastase nor with cathepsin G. NP4 was purified and electrophoresis of the affinity-purified enzyme in sodium dodecyl sulfate polyacrylamide gels resulted in a single Mr = 30,000 polypeptide. The purified enzyme digested fibrin but not elastin and it cleaved Boc-Ala-ONp readily (Km = 0.47mM) at neutral pH, but had no effect on Suc-[Ala]3 Nan and N-Suc-[Ala]2-Pro-Phe-pNA. The proteolytic activity was inhibited by DFP, alpha 1 PI and alpha 2 M with a Ki of 10(-9)M for the NP4-alpha 1 PI complex. The NH2-terminal sequence and the amino-acid composition of NP4 were distinct from those of elastase and cathepsin G. Neutrophils contain large amounts of NP4 as judged by the comparable amounts of elastase- and NP4-alpha 1 PI complexes present in inflammatory exudates.     

Inhibitors of elastase and cathepsin G in Chédiak-Higashi (beige) neutrophils

Previous studies have established that mature neutrophils from the peritoneal cavity, blood, and bone marrow of beige (Chédiak-Higashi syndrome) mice essentially lack activities of two lysosomal proteinases: elastase and cathepsin G. There are, however, significant levels of each enzyme in early neutrophil precursors in bone marrow. In the present experiments, it was found that the addition of extracts from mature beige neutrophils to extracts of normal neutrophils or to purified human neutrophil elastase and cathepsin G resulted in a significant inhibition of elastase and cathepsin G G activities. 125I-Labeled human neutrophil elastase formed high molecular mass complexes at 64 and 52 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis when added to beige neutrophil extracts. The molecular masses of the inhibitor-125I-elastase complexes suggested that the molecular masses of the inhibitors are approximately 36 and 24 kDa, respectively. These results were confirmed by gel filtration on Superose 12 under nondenaturing conditions. Cathepsin G was inhibited only by the 36-kDa component. The inhibitors formed a covalent complex with the active sites of elastase and cathepsin G. No inhibitory activity was present in mature neutrophil extracts of genetically normal mice or in extracts of bone marrow of beige mice. These results thus represent an unusual example of an enzyme deficiency state caused by the presence of excess inhibitors. Inactivation of neutrophil elastase and cathepsin G in mature circulating and tissue neutrophils may contribute to the increased susceptibility of Chédiak-Higashi patients to infection.     

Secreted forms of human neutrophil collagenase

Collagenase in human neutrophils is found within intracellular granules which can be stimulated to be secreted with phorbol myristic acetate. This extracellular secreted form of neutrophil collagenase was isolated by immunoaffinity chromatography using a monoclonal antibody previously shown to specifically recognize neutrophil collagenase. The enzyme efficiently bound to this column and was eluted with NaSCN as three major species of 75, 57, and 22 kDa, respectively. These proteins were closely related immunologically since, after radiolabeling and separation by gel filtration, each of the three proteins was precipitated by the monoclonal antibody. Also, the 75- and 57-kDa proteins exhibited collagenase activity after elution from polyacrylamide gels run under nondenaturing conditions. Further, the 57-kDa protein autodegraded into a 22-kDa protein with time. Polyclonal antibody, prepared to the 57-kDa enzyme, also recognized the 75- and 22-kDa proteins using an immunoblot technique. When crude neutrophil supernatants containing latent collagenase were immunoblotted, both the 75- and the 57-kDa enzymes were present. Our immunoaffinity purified active enzymes, although activated during the course of purification, resemble the latent enzymes in crude neutrophil supernatants. The multiple forms of secreted collagenase from degranulated leukocytes may resemble more closely that seen in inflammation.     

Enzymatic and Molecular Biochemical Characterizations of Human Neutrophil Elastases and a Cathepsin G-like Enzyme

Human neutrophil elastase (HNE, EC 3. 4. 21. 37) is a causative factor of inflammatory diseases, including emphysema and rheumatoid arthritis. Enzymatic characterization is important for the development of new drugs involved in the regulation of this enzyme. In this study, we investigated the enzymatic and biochemical properties of five different elastolytic enzymes, with a molecular mass between 24 kDa and 72 kDa. Three elastases, molecular masses of 27, 29, 31 kDa, might be elastase isozymes that have the same NH₂-terminal amino acid sequences of Ile-Val-Gly-Gly-Arg-Arg-Ala. The 24-kDa enzyme, which showed the identical NH₂-terminal amino acid sequences to elastase, was a degraded fragment of native elastase. The elastolytic activity was conserved at the 6/7 domain of the NH₂-terminal region. The inhibitory characteristics of PMSF, DipF were the same as those of native elastases. The 72-kDa molecule, which showed elastolytic activity, might be a trimer formed between native elastases (31 kDa and 29 kDa) and a cathepsin G-like enzyme, which did not show elastolytic activity but enhanced the elastolytic activity of neutrophil elastase. Although this cathepsin G-like enzyme showed weak cathepsin G activity, it has distinguishable NH₂-terminal sequences of Ile-Val-Gly-Gly-Ser-Arg-Ala- from those of elastase or cathepsin G. The potentiation of elastolytic activity could be a result of the trimerization of native elastase with a cathepsin G-like enzyme, and was then weakly inhibited by serine protease inhibitors, such as PMSF, DipF. Therefore, we suggest the cathepsin G-like enzyme to be a novel enzyme, which has an important role in the development of inflammation.     

Solar Ultraviolet Irradiation Induces Decorin Degradation in Human Skin Likely via Neutrophil Elastase

Exposure of human skin to solar ultraviolet (UV) irradiation induces matrix metalloproteinase-1 (MMP-1) activity, which degrades type I collagen fibrils. Type I collagen is the most abundant protein in skin and constitutes the majority of skin connective tissue (dermis). Degradation of collagen fibrils impairs the structure and function of skin that characterize skin aging. Decorin is the predominant proteoglycan in human dermis. In model systems, decorin binds to and protects type I collagen fibrils from proteolytic degradation by enzymes such as MMP-1. Little is known regarding alterations of decorin in response to UV irradiation. We found that solar-simulated UV irradiation of human skin in vivo stimulated substantial decorin degradation, with kinetics similar to infiltration of polymorphonuclear (PMN) cells. Proteases that were released from isolated PMN cells degraded decorin in vitro. A highly selective inhibitor of neutrophil elastase blocked decorin breakdown by proteases released from PMN cells. Furthermore, purified neutrophil elastase cleaved decorin in vitro and generated fragments with similar molecular weights as those resulting from protease activity released from PMN cells, and as observed in UV-irradiated human skin. Cleavage of decorin by neutrophil elastase significantly augmented fragmentation of type I collagen fibrils by MMP-1. Taken together, these data indicate that PMN cell proteases, especially neutrophil elastase, degrade decorin, and this degradation renders collagen fibrils more susceptible to MMP-1 cleavage. These data identify decorin degradation and neutrophil elastase as potential therapeutic targets for mitigating sun exposure-induced collagen fibril degradation in human skin.     

Solid-phase radioimmunoassay for human neutrophil elastase: a sensitive method for determining secreted and cell-associated enzyme

A solid-phase radioimmunoassay for measuring neutrophil elastase in the range 0.08-4 ng/ml has been developed. A monospecific, precipitating antibody capable of inhibiting elastinolysis was produced by repeated immunizations of a goat. The IgG fraction and affinity-purified antibodies of this serum were then obtained and used to develop this radioimmunoassay. There was no cross-reactivity in binding of the radiolabeled antisera with lactoferrin, cathepsin G, or serine proteinases with amino-terminal amino acid sequence homology. Although serum influences the measurement of catalytically active neutrophil elastase when compared to diisopropylfluorophosphate-treated neutrophil elastase, antigenic elastase may still be measured in body fluids. Furthermore, this assay is more sensitive than commercially available substrates used for quantitating neutrophil elastase by functional activity. We have found this quantitative assay extremely useful in balance studies to measure secreted and cell-associated elastase and in screening of biological fluids for the presence of the enzyme.     

Solid-phase immunoassay of dog neutrophil elastase

A sensitive and reliable method has been developed for the detection of dog neutrophil elastase using the amplified enzyme-linked immunosorbent assay. Purified neutrophil enzyme, inactivated by diisopropylfluorophosphate was adsorbed noncovalently to polystyrene tubes (11 × 55 mm) and immune rabbit serum was allowed to bind to antigen-sensitized tubes. Bound specific antibody was visualized by goat antirabbit immunoglobulin covalently lined to alkaline phosphatase, using p-nitrophenyl phosphate as the substrate. Increasing amounts of purified neutrophil enzyme or crude leukocyte extracts were quantitated by their ability to inhibit specific antibody uptake to polystyrene tubes. By this method, as little as 1 ng of enzyme/ml could be detected with a useful range of 5–100 ng of enzyme levels. Immunoreactive enzyme in a crude leukocyte extract was comparable to the quantily of enzyme as measured by proteolytic activity. The method described can be conveniently used to measure levels of immunoreactive enzymes in biological fluids of animals, with experimentally induced inflammatory conditions.     

Susceptibility of cartilage collagens type II, IX, X, and XI to human synovial collagenase and neutrophil elastase

The action of purified rheumatoid synovial collagenase and human neutrophil elastase on the cartilage collagen types II, IX, X and XI was examined. At 25 degrees C, collagenase attacked type II and type X (45-kDa pepsin-solubilized) collagens to produce specific products reflecting one and at least two cleavages respectively. At 35 degrees C, collagenase completely degraded the type II collagen molecule to small peptides whereas a large fragment of the type X molecule was resistant to further degradation. In contrast, collagen type IX (native, intact and pepsin-solubilized type M) and collagen type XI were resistant to collagenase attack at both 25 degrees C and 35 degrees C even in the presence of excess enzyme. Mixtures of type II collagen with equimolar amounts of either type IX or XI did not affect the rate at which the former was degraded by collagenase at 25 degrees C. Purified neutrophil elastase, shown to be functionally active against soluble type III collagen, had no effect on collagen type II at 25 degrees C or 35 degrees C. At 25 degrees C collagen types IX (pepsin-solubilized type M) and XI were also resistant to elastase, but at 35 degrees C both were susceptible to degradation with type IX being reduced to very small peptides. Collagen type X (45-kDa pepsin-solubilized) was susceptible to elastase attack at 25 degrees C and 35 degrees C as judged by the production of specific products that corresponded closely with those produced by collagenase. Although synovial collagenase failed to degrade collagen types IX and XI, all the cartilage collagen species examined were degraded at 35 degrees C by conditioned culture medium from IL1-activated human articular chondrocytes. Thus chondrocytes have the potential to catabolise each cartilage collagen species, but the specificity and number of the chondrocyte-derived collagenase(s) has yet to be resolved.     

Characterization of alkaline phosphatase from bovine polymorphonuclear neutrophils

We characterized the bovine polymorphonuclear neutrophil alkaline phosphatase which was considerably purified with a sp. act. of 206 units/mg of protein. The Km value for p-nitrophenylphosphate at pH 10.0 was 1.69 mM. L-Histidine, imidazole and L-homoarginine but not L-phenylalanine inhibited the enzyme. In heat stability study, the enzyme lost 50% activity at 56 degrees C for 20 min. The enzyme had a half-life of 30 min in 3 M urea at 37 degrees C and pH 7.5. The enzyme was inhibited by beta-mercaptoethanol in a dose-dependent fashion. It is suggested from above results that the neutrophil alkaline phosphatase isozyme could be distinguishable from other tissue isozymes.     

The latent collagenase and gelatinase of human polymorphonuclear neutrophil leucocytes.

Two metallo-proteinases of human neutrophil leucocytes, collagenase and gelatinase, were studied. Collagenase specifically cleaved native collagen into the TCA and TCB fragments, whereas gelatinase degraded denatured collagen, i.e. gelatin, and the TCA fragments produced by collagenase. On subcellular fractionation by zonal sedimentation, collagenase was found to be localized in the specific granules, separate from gelatinase, which was recovered in smaller subcellular organelles known as C-particles. Neither enzyme was present in the azurophil granules, which contain the two major serine proteinases of neutrophils, elastase and cathepsin G. Collagenase and gelatinase were separated by gel filtration from extracts of partially purified granules. Both enzymes were found to occur in latent forms and were activated either by trypsin or by 4-aminophenylmercuric acetate. Gelatinase was also activated by cathepsin G, which, however, destroyed collagenase. Both enzymes were destroyed by neutrophil elastase. Activation resulted in a decrease by 25 000 in the apparent mol. wt. of both latent metallo-proteinases.     

Corticosteroid-binding globulin reactive centre loop antibodies recognise only the intact natured protein: elastase cleaved and uncleaved CBG may coexist in circulation

Corticosteroid-binding globulin (CBG) is the principal carrier of cortisol in circulation and is a non-inhibitory member of the serpin family of serine proteinase inhibitors. It possesses an exposed elastase specific site which, when cleaved, allows a conformational change promoting the delivery of cortisol to sites of inflammation. Previously there was no ability to independently distinguish between the uncleaved, stressed, conformer of CBG and total CBG in circulation. Here we raised and characterized monoclonal antibodies generated against a synthetic peptide spanning the elastase cleavage site within the exposed reactive centre loop (RCL) and measured changes in CBG by ELISA following treatment with human neutrophil elastase. The antibodies recognized the synthetic peptide as well as intact CBG and the epitope (STGVTLNL) spanned the elastase cleavage site. Treatment of plasma with elastase resulted in a complete loss of CBG levels determined using these RCL antibodies whereas CBG levels measured with an unrelated CBG monoclonal antibody were unaffected. We also compared plasma levels of CBG measured by RCL antibodies and an unrelated CBG antibody and showed discordance in some samples. This study shows for the first time the ability to measure the intact, stressed conformer of CBG. We report discordance with total CBG in some samples implying the presence of cleaved CBG in circulation. This is an important finding as it has implications for free cortisol which hitherto have been determined from total cortisol and total CBG levels. This antibody could be used for determining the time course of intact CBG in various relevant patient cohorts and for structure/function studies on the biology of human CBG.     

Characterization of elastase associated with granulomatous tissue remodeling

Elastases have been reported to be involved in various types of tissue injury. In this study we detected hydrolytic activities for [3H]-elastin and Suc-Ala-Ala-Ala-pNA (SLAPN) in hepatic granulomas which became elevated in parallel with enlargement of the granulomas and disappearance of aldehyde-fuchsin-stained filaments in the lesions of mice infected with Schistosoma mansoni. The elastase was partially purified by gel filtration followed by anion-exchange chromatography. This enzyme has a molecular weight of 20-25k and hydrolyzed denatured collagen (azocoll), Glu-Pro-Val-pNA, SLAPN, and [3H]-elastin. Optimal pH was 7-8.5. It is a serine proteinase and distinct in its inhibitor profile from murine peritoneal macrophage elastase, which has been reported by others. Digestion of elastic fibers in vessel walls and fine fibrils in newly developed granulomas by the granuloma elastase was histochemically identified with aldehyde-fuchsin stain. These results indicate that a serine proteinase functions as a major elastase in granulomatous tissue remodeling and may account for the disappearance of elastic fibers and other elements of the matrix in fully developed granulomas.     

Human specific anti-type IV collagen monoclonal antibodies, characterization and immunohistochemical application

This paper describes two new monoclonal antibodies reactive with human specific type IV collagen epitopes in frozen as well as routinely fixed and processed tissue sections. The antibodies (1042 and 1043) were raised against human placental type IV collagen and were shown by immunoblotting and ELISA tests to react exclusively with type IV collagen determinants. Extensive immunohistochemical survey studies on panels of tissues from various species, using unfixed cryostat sections, demonstrated that antibody 1042 reacted only with human type IV collagen whereas antibody 1043 in addition reacted with rabbit type IV collagen. All tissues showed homogeneous staining of the basement membrane, indicating that the detected epitopes did not show organ-specific distribution. Tissue processing protocols for using these monoclonal antibodies on routinely processed paraffin embedded tissues were developed. It was found that whereas polyclonal anti-type IV collage antisera required pepsin digestion, our monoclonal antibodies required pronase or papain digestion to restore type IV collagen immunoreactivity in paraffin sections. It is concluded that these monoclonal anti-type IV collagen antibodies detect species specific epitopes which can be detected in routinely processed paraffin embedded tissues after appropriate enzyme pretreatment.     

Variations in elastaselike esterase activities in human leucocytes during cell maturation.

Granules of human peripheral blood leucocytes contain four well-characterized elastase isozymes and one or two slow-moving elastaselike esterases (SE) which have not been as well characterized. SE are capable of hydrolyzing typical elastase synthetic sybstrates such as N-acetyl-dl-alanine-alpha-naphthyl ester (Ac-DL-Ala-1-ONap) and N-t-butyloxycarbonyl-L-alanine-p-nitrophenyl ester (Boc-Ala-ONp), but unlike the highly basic elastase isozymes, SE barely migrate into 13% acrylamide gels during cationic electrophoresis at pH 4.3. Hydrolysis of Ac-DL-Ala-1-ONap by SE requires the presence of Triton in the gel, and hydrolysis of Boc-Ala-ONp by the same enzyme(s) is also enhanced in the presence of the detergent. Triton is not required for these activities, in the case of the elastase isozymes. Diisopropylfluorophosphate (Dip-F) inactivates both SE and the elastase isozymes, whereas Ac-(Ala)2-Pro-AlaCH2Cl (a powerful inactivator of the leucocyte elastase isozymes at 10-4 M concentration) does not inactivate SE at the same concentration. Immunochemical studies revealed antigenic cross-reaction between the rapidly migrating leucocyte elastase isozymes and SE. Two preparations of leucocyte granules from nonleukemic bone marrow cells showed no activity of the rapidly migrating elastase isozymes, but did contain SE activity. SE may be a precursor or zymogen form of the elastase isozymes, present in immature cells and partly retained through later stages of development.     

Production and characterization of a monoclonal antibody to human type III collagen

Human type III collagen from placenta was isolated and purified for use as an immunogen. A monoclonal antibody was produced which specifically recognizes epitopes unique to type III collagen. The specificity of the antibody was determined by inhibition ELISA, an immunoblot assay, and by immunoprecipitation. Results indicated that the monoclonal antibody recognized only the alpha 1(III) polypeptide chains and did not crossreact with type I, IV, or V collagen. The monoclonal antibody was also used for immunohistochemical localization of type III collagen in tissue sections of human placenta, bovine spleen, and lymph node. In placenta, both large and small blood vessels showed pronounced staining of the tunica media, which contains largely smooth muscle cells, known to synthesize type III collagen. In contrast, the intimal areas and endothelial cells showed no staining with the antibody. In the placental villi, staining was limited to the villous core, where fine fibrillar structures showed strong staining. In lymph nodes, the capsule and pericapsular adipose cells were surrounded by a covering of type III collagen. Within the parenchyma of the node, staining was localized to a branching, reticular array of fine fibers. In the spleen, staining was pronounced in the capsule, splenic trabeculae, and white pulp, where blood vessel staining was especially prominent. The red pulp and splenic sinuses contain little or no type III collagen. The fine network-like or reticular staining pattern found in the lymph node parenchyma is consistent with the staining pattern of the protein reticulin, and suggests that type III collagen may be closely associated with reticulin in certain tissues. Since the role of type III in tissues is unclear, this reagent will be useful in providing new information in this regard.     

Cleavage of type VIII collagen by human neutrophil elastase.

In this report, the susceptibility of type VIII collagen to human neutrophil elastase is compared to other extracellular matrix components. Type X collagen is degraded to specific fragments at a substrate to enzyme ratio of 5:1 after 20 h at room temperature, but type VIII collagen is almost completely degraded after only 4 h incubation at a substrate to enzyme ratio of 50:1 and partly degraded after only 15 min. Laminin, merosin and types I, III, IV and V collagen exhibit no susceptibility to neutrophil elastase under the latter conditions, while fibronectin is degraded.     

Purification and characterization of a novel cysteine proteinase (periodontain) from Porphyromonas gingivalis. Evidence for a role in the inactivation of human alpha1-proteinase inhibitor

Periodontal disease is characterized by inflammation of the periodontium manifested by recruitment of neutrophils, which can degranulate, releasing powerful proteinases responsible for destruction of connective tissues, and eventual loss of tooth attachment. Although the presence of host proteinase inhibitors (serpins) should minimize tissue damage by endogenous proteinases, this is not seen clinically, and it has been speculated that proteolytic inactivation of serpins may contribute to progression of the disease. A major pathogen associated with periodontal disease is the Gram-negative anaerobe Porphyromonas gingivalis, and in this report, we describe a novel proteinase that has been isolated from culture supernatants of this organism that is capable of inactivating the human serpin, alpha1-proteinase inhibitor, the primary endogenous regulator of human neutrophil elastase. This new enzyme, referred to as periodontain, belongs to the cysteine proteinase family based on inhibition studies and exists as a 75-kDa heterodimer. Furthermore, periodontain shares significant homology to streptopain, a proteinase from Streptococcus pyogenes, and prtT, a putative proteinase from P. gingivalis. Clearly, the presence of this enzyme, which rapidly inactivates alpha1-proteinase inhibitor, could result in elevated levels of human neutrophil elastase clinically detected in periodontal disease and should be considered as a potential virulence factor for P. gingivalis.     

Molecular cloning of complementary DNA encoding one of the human pancreatic protease E isozymes

We have cloned a DNA from a human pancreatic cDNA library using a cloned rat pancreatic elastase 1 cDNA as a probe, and determined its nucleotide sequence. This cDNA contains a coding region of 810 nucleotides which encodes a 270-amino-acid protein. The deduced amino acid sequence shows less than 60% homologies with rat and porcine pancreatic elastase 1, although its substrate binding region is homologous with those of the above elastases 1. When this deduced amino acid sequence was compared with known amino acid sequences of pancreatic proteases other than elastases, it was found to contain an amino acid sequence which was highly homologous with the N-terminal amino acid sequence of porcine pancreatic protease E. We also purified human pancreatic protease E isozymes from human pancreatic juice, and determined their N-terminal amino acid sequences. One of the isozymes does not hydrolyze elastin but does hydrolyze a synthetic substrate. Endoglycosidase F digests glycoside bonds of the isozyme. These results suggest that the cDNA cloned by us corresponded to one of the human protease E isozymes.     

Low-dose chymotrypsin treatment inhibits neutrophil migration into sites of inflammation in vivo: effects on Mac-1 and MEL-14 adhesion protein expression and function

Antibody blocking studies in the mouse suggest that the MEL-14 antigen is involved in neutrophil-endothelial cell interactions and may be important in neutrophil extravasation to sites of inflammation in vivo. We recently showed that chemotactic factor activation causes a rapid (within minutes) shedding of a large fragment of the MEL-14 antigen from the surface of neutrophils. We report here that chymotrypsin, at low doses (0.1 units/1 x 10(6) cells), but not trypsin, elastase, or collagenase, causes an activation-independent rapid loss (greater than 90%) of the MEL-14 antigen from the surface of murine neutrophils. Under the same treatment conditions chymotrypsin has no effect on the expression of four other neutrophil surface antigens, including the Mac-1 adhesion protein. Chymotrypsin treatment has no effect on neutrophil adhesion to plastic, migration to C5a, regulation of the Mac-1 antigen, but causes a greater than 95% reduction in neutrophil binding to high endothelial venules (HEV) in peripheral lymph nodes measured in the ex vivo frozen section HEV binding assay. The level of inhibition of neutrophil adhesion to HEV was comparable to that seen with the MEL-14 antibody. This experimental system allows us for the first time to specifically examine the consequences of removing the MEL-14 antigen from the surface of neutrophils on function in vivo. We show that treatment with chymotrypsin blocks greater than 85% of the ability of neutrophils injected back into the animal to home to the inflamed peritoneum. In similar in vivo experiments the MEL-14 antibody blocks neutrophil homing by 60-70%. These results further support the importance of the MEL-14 antigen in neutrophil extravasation in vivo and indicate that chymotrypsin could be useful in examining the molecular mechanisms involved in extravasation of leukocytes into a variety of diverse tissue sites of inflammation.