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Neutrophil granulocytes form the body's first line of antibacterial defense, but they also contribute to tissue injury and noninfectious, chronic inflammation. Proteinase 3 (PR3) and neutrophil elastase (NE) are 2 abundant neutrophil serine proteases implicated in antimicrobial defense with overlapping and potentially redundant substrate specificity. Here, we unraveled a cooperative role for PR3 and NE in neutrophil activation and noninfectious inflammation in vivo, which we believe to be novel. Mice lacking both PR3 and NE demonstrated strongly diminished immune complex-mediated (IC-mediated) neutrophil infiltration in vivo as well as reduced activation of isolated neutrophils by ICs in vitro. In contrast, in mice lacking just NE, neutrophil recruitment to ICs was only marginally impaired. The defects in mice lacking both PR3 and NE were directly linked to the accumulation of antiinflammatory progranulin (PGRN). Both PR3 and NE cleaved PGRN in vitro and during neutrophil activation and inflammation in vivo. Local administration of recombinant PGRN potently inhibited neutrophilic inflammation in vivo, demonstrating that PGRN represents a crucial inflammation-suppressing mediator. We conclude that PR3 and NE enhance neutrophil-dependent inflammation by eliminating the local antiinflammatory activity of PGRN. Our results support the use of serine protease inhibitors as antiinflammatory agents.

1. Mediators of inflammation can increase vascular permeability in at least two different ways: by acting directly on endothelial cells or, indirectly, through an incompletely understood mechanism, dependent on circulating neutrophils. Neutrophil-dependent oedema formation has been described in the skin of rabbits, rats, hamsters, mice and man. In contrast, we presented evidence in a previous study that local oedema formation induced by i.d. injection of chemoattractants in guinea-pig skin was neutrophil-independent. In the present study, we sought evidence of neutrophil-dependent oedema formation in immune-complex-mediated vasculitis, the reversed passive Arthus (RPA) reaction, in guinea-pig skin. We also investigated whether haemorrhage in the RPA reaction was neutrophil-dependent (as it is in other species) and the role of endogenous mediators of inflammation (prostaglandins, nitric oxide, histamine, PAF and leukotrienes) in contributing to the local inflammatory response. 2. In the RPA reaction, most oedema formation occurred over the first 60 min whereas 111In-neutrophil accumulation was still increasing from 60 to 240 min. The different kinetics of these two events suggested that they may be dissociated. 3. Oedema formation was partially inhibited by a long-acting PAF antagonist (UK-74,505) and an H1 histamine receptor antagonist (mepyramine) but not by a 5-lipoxygenase inhibitor (ZM 230487). A nitric oxide synthesis inhibitor (NG-nitro-L-arginine methyl ester, L-NAME) suppressed oedema formation by 68% whereas a cyclo-oxygenase inhibitor suppressed oedema by 27%. 4. 111In-neutrophil accumulation in the RPA reaction was partially suppressed by UK-74,505. In contrast, ZM 230487 was without effect at doses which abrogated arachidonic acid-induced 111In-neutrophil accumulation. 5. The anti-CD18 monoclonal antibody, (mAb) 6.5E F(ab')2, effectively inhibited 111In-neutrophil accumulation induced by PAF, zymosan-activated plasma (ZAP) and in the RPA reaction. However, oedema formation measured in the same sites was not altered. In contrast, oedema formation in the RPA reaction was partially suppressed by 6.5E whole mAb which was 2.5 times more potent than 6.5EF(ab')2 at inhibiting guinea-pig neutrophil adhesion to protein-coated plastic. Haemorrhage induced by PAF and in the RPA reaction was significantly inhibited by 6.5E F(ab')2 pretreatment.6. We conclude that in the RPA reaction in guinea-pig skin, oedema formation is partially neutrophil dependent as assessed by using an anti-CD18 mAb, whereas ZAP-induced oedema formation is neutrophil-independent. Haemorrhage was also dependent on neutrophil accumulation. In addition, our studies support a role for PAF in mediating both oedema formation and "'In-neutrophil accumulation in the RPA reaction. Endogenous release of histamine also appears to be important in mediating oedema formation suggesting that mast cells play a critical role in increases of vascular permeability in inflammatory reactions in guinea-pig skin. Moreover, our results confirm previous findings which suggest a dominant role for nitric oxide in maintaining cutaneous blood flow in the guinea-pig.

Cutaneous late phase reactions (LPR) in rats can be induced by the intradermal injection of anti-IgE antibody or isolated rat peritoneal mast cell granules. Rat LPR are characterized by neutrophil-rich infiltrates at 2 to 8 hr followed by mononuclear cell-rich infiltrates thereafter. Rat Arthus reactions are histologically similar and are complement (C) dependent. To determine the importance of C in the pathogenesis of rat LPR compared with its role in Arthus reactions, rats were treated with cobra venom factor (CVF) (250 U/kg i.v.), and the effects of this treatment on total hemolytic complement (CH50), C3 titers, LPR, and Arthus reactions were assessed. CVF treatment produced profound decreases in both CH50 (from 197 +/- 20 to less than 1.0 U/ml) and C3 (from 44,240 +/- 2840 to less than 5 U/ml) titers after 6 hr, which persisted through at least 30 hr. The inflammatory intensity of heterologous reverse passive Arthus reactions was significantly decreased in CVF-treated animals. In contrast, the intensity of LPR was unaffected by CVF treatment. Therefore, although LPR and Arthus reactions share certain histologic characteristics, these similarities are not due to a mutual requirement for the presence of C.

The generation of toxic oxygen products from activated phagocytic cells represents an important pathogenic mechanism in tissue damage associated with inflammatory reactions that are characterized by the involvement of neutrophils or other phagocytic cells. The production of these toxic products from activated phagocytic cells is related to receptor activation on the surface of the cells. Most emphasis has been placed on studies in the lung where the production of these toxic metabolites by phagocytic cells, either in the vasculature or in the airway compartment of the lungs, has been demonstrated to result in endothelial cell injury or damage and destruction of alveolar lining cells. In some cases these reactions are progressive, resulting in parenchymal collapse and fibrosis. Naturally occurring protective factors have been demonstrated both within cells and in the plasma and interstitial fluid compartments. Although there is increasing evidence that in the shock syndrome complement activation products and toxic oxygen metabolites from activated leukocytes play important roles, the most direct evidence for related mechanisms comes in detailed studies of immune complex induced injury where a role for both O-2 and H2O2 has been clearly demonstrated. These facts indicate that toxic oxygen metabolites from activated phagocytic cells play important roles in a variety of pathological situations, and have provided new insights into intervention therapy.

The ability of antisera to guinea-pig C3 to inhibit the Arthus and local Shwartzman reactions was studied. They were found to reduce the non-haemorrhagic component of the active and reversed passive Arthus reactions and to delay the appearance of the haemorrhage in the active Arthus reaction. Cobra venom factor, however, had no effect on the non-haemorrhagic components of these reactions and only delayed the appearance of the haemorrhage of the active Arthus reaction. There appeared to be a correlation between the serum complement level and the time taken for the haemorrhage to appear, and between the circulating platelet count and the extent of the non-haemorrhagic, oedematous component of the reaction. The haemorrhagic component of the local Shwartzman reaction was not affected by decomplementation with cobra venom factor. The ability of the antisera to inhibit the haemorrhage of the Shwartzman reaction was not dependent on lowering the serum complement titre. However, the haemorrhage was inhibited if the circulating platelet count was also reduced to very low numbers. Antiserum to zymosan alone had the same effect as anti-beta1C/beta1A globulin (zymosan) in blocking the reaction, although it did not alter the complement levels or the platelet counts. The possibility of an immunological cross-reactivity between zymosan and endotoxin in this action is discussed.

An experimental model was utilized to examine the physiological effects of nonsteroidal anti-inflammatory compounds on locally induced inflammatory lesions in laboratory rabbits. The modulation of specific parameters associated with the inflammatory response, was monitored in vivo using radiolabeled cells and proteins, following the local administration of either indomethacin (INDO), acetylsalicylic acid (ASA) or sterile saline (as control) in the dermis. In one half of the dorsal aspect of each animal, Arthus-type dermal lesions were induced in triplicate, while similar sites were prepared at identical time periods (1-4, and 6 h) on the contralateral halves. In these latter sites, either INDO or ASA was administered (100 micrograms/site). The non-drug-treated lesions served as intrinsic controls when the inflammatory parameters of vascular permeability, neutrophil (polymorphonuclear) leukocyte infiltration and hemorrhage were investigated. Also, sterile saline or drug-injected, noninflamed sites prepared in each half served as further controls. Statistical analysis indicated a significant suppression of the above-named inflammatory parameters when either INDO or ASA was applied to localized inflammatory sites, as compared to non-drug-treated lesions. This study may serve to illustrate the value of deploying nonsteroidal anti-inflammatory preparations, topically, when conservatively managing the acute patient in clinical practice.

The aims of the studies presented in this publication were to elucidate the morphology and quantitate the kinetics of an inflammatory reaction elicited by immune complexes and to ascertain the role of complement in the reaction. The hallmark of both the direct active (DAA) and reversed passive (RPA) Arthus reactions was the accumulation of immune precipitates and polymorphonuclear leukocytes (PMNs) in and around vessels. Using fluoresceinated antigen as a tracer, immune complexes localized in the lumina and walls of venules and small veins in the DAA and in the wall of vessels and perivascularly in RPA. PMNs accumulated at these same sites, phagocytosed the fluoresceinated complexes and became degranulated. The precise localization of immune complexes was achieved by examining the same tissue sections first by fluorescence microscopy, followed by conventional staining and examination by light microscopy. Marked stasis of the microcirculation was observed, particularly in DAA, in which a few immune complex-containing PMNs were entrapped in a mass of densely packed red blood cells. Some edema was observed in early lesions and definitive separation of collagen fibers was noted in lesions older than 2 hr. Hemorrhage became the dominant characteristic of both types of reactions from 2 hr onward. By administering radiolabeled cells, proteins, and microspheres as a "pulse," given at various times before sacrifice, the quantitation and kinetics of the inflammatory lesions elicited by immune complexes could be elucidated. In RPA all parameters quantitated reached a peak soon after elicitation of the reaction (2-4 hr), which is in keeping with other forms of acute inflammation. In DAA there was some difficulty in assessing the quantitation because of interanimal variations and because of progression of the inflammatory lesions, as the antigen diffused peripherally from the site of its injection. Peak activities occurred in 4- to 8-hr-old lesions. These observations and a comparison of the center and periphery of the lesions, strengthen the contention that the RPA and DAA have common features and features which differ. In common are immunological mechanisms (antigen-antibody interaction and complement activation) and cellular events (polymorphonuclear leukocyte chemotaxis, phagocytosis, and release of lysosomal contents). Different features are the site of immune complex formation and its sequelae. In RPA they form primarily in the wall of venules and small veins and hence have a marked effect on increase in vessel permeability. In the DAA most of the complexes form and the leukocytes accumulate in the lumen of the same vessels.(ABSTRACT TRUNCATED AT 400 WORDS)