Group X secretory PLA2 in neutrophils plays a pathogenic role in abdominal aortic aneurysms in mice

Group X secretory PLA(2) (sPLA(2)-X) is expressed in neutrophils and plays a role in the pathogenesis of neutrophil-mediated tissue inflammation and injury. This study tested the hypothesis that sPLA(2)-X in neutrophils may contribute to the pathogenesis of abdominal aortic aneurysms (AAA) using sPLA(2)-X(-/-) mice. AAA was created by application of CaCl(2) to external surface of aorta. As a result, the aortas of sPLA(2)-X(-/-) mice had smaller diameters (percent increase from baseline; 24.8 ± 3.5% vs. 49.9 ± 9.1%, respectively; P < 0.01), a reduced grade of elastin degradation, and lower activities of elastase and gelatinase (26% and 19% lower, respectively) after CaCl(2) treatment compared with sPLA(2)-X(+/+) mice. In sPLA(2)-X(+/+) mice, immunofluorescence microscopic images showed that the immunoreactivity of sPLA(2)-X was detected only in neutrophils within aortic walls 3 days, 1, 2, and 6 wk after CaCl(2) treatment, whereas the immunoreactivity was not detected in macrophages or mast cells in aortic walls. sPLA(2)-X immunoreactivity also was colocalized in cells expressing matrix metalloproteinase (MMP)-9. Neutrophils isolated from sPLA(2)-X(-/-) mice had lower activities of elastase, gelatinase, and MMP-9 in response to stimuli compared with sPLA(2)-X(+/+) mice. The attenuated release of elastase and gelatinase from sPLA(2)-X(-/-) neutrophils was reversed by exogenous addition of mouse sPLA(2)-X protein. The adoptive transfer of sPLA(2)-X(+/+) neutrophils days 0 and 3 after CaCl(2) treatment reversed aortic diameters and elastin degradation grades in the lethally irradiated sPLA(2)-X(+/+) mice reconstituted with sPLA(2)-X(-/-) bone marrow to an extent similar to that seen in sPLA(2)-X(+/+) mice. In conclusion, sPLA(2)-X in neutrophils plays a pathogenic role in AAA in a mice model.     

Anthrax toxin targeting of myeloid cells through the CMG2 receptor is essential for establishment of Bacillus anthracis infections in mice

Bacillus anthracis kills through a combination of bacterial infection and toxemia. Anthrax toxin working via the CMG2 receptor mediates lethality late in infection, but its roles early in infection remain unclear. We generated myeloid-lineage specific CMG2-deficient mice to examine the roles of macrophages, neutrophils, and other myeloid cells in anthrax pathogenesis. Macrophages and neutrophils isolated from these mice were resistant to anthrax toxin. However, the myeloid-specific CMG2-deficient mice remained fully sensitive to both anthrax lethal and edema toxins, demonstrating that targeting of myeloid cells is not responsible for anthrax toxin-induced lethality. Surprisingly, the myeloid-specific CMG2-deficient mice were completely resistant to B. anthracis infection. Neutrophil depletion experiments suggest that B. anthracis relies on anthrax toxin secretion to evade the scavenging functions of neutrophils to successfully establish infection. This work demonstrates that anthrax toxin uptake through CMG2 and the resulting impairment of myeloid cells are essential to anthrax infection.     

Anthrax lethal toxin has direct and potent inhibitory effects on B cell proliferation and immunoglobulin production

Protective host immune responses to anthrax infection in humans and animal models are characterized by the development of neutralizing Abs against the receptor-binding anthrax protective Ag (PA), which, together with the lethal factor (LF) protease, composes anthrax lethal toxin (LT). We now report that B cells, in turn, are targets for LT. Anthrax PA directly binds primary B cells, resulting in the LF-dependent cleavage of the MAPK kinases (MAPKKs) and disrupted signaling to downstream MAPK targets. Although not directly lethal to B cells, anthrax LT treatment causes severe B cell dysfunction, greatly reducing proliferative responses to IL-4-, anti-IgM-, and/or anti-CD40 stimulation. Moreover, B cells treated with anthrax LT in vitro or isolated from mice treated with anthrax LT in vivo have a markedly diminished capacity to proliferate and produce IgM in response to TLR-2 and TLR-4 ligands. The suppressive effects of anthrax LT on B cell function occur at picomolar concentrations in vitro and at sublethal doses in vivo. These results indicate that anthrax LT directly inhibits the function of B cells in vitro and in vivo, revealing a potential mechanism through which the pathogen could bypass protective immune responses.     

Inflammasome sensor Nlrp1b-dependent resistance to anthrax is mediated by caspase-1, IL-1 signaling and neutrophil recruitment

Bacillus anthracis infects hosts as a spore, germinates, and disseminates in its vegetative form. Production of anthrax lethal and edema toxins following bacterial outgrowth results in host death. Macrophages of inbred mouse strains are either sensitive or resistant to lethal toxin depending on whether they express the lethal toxin responsive or non-responsive alleles of the inflammasome sensor Nlrp1b (Nlrp1b(S/S) or Nlrp1b(R/R), respectively). In this study, Nlrp1b was shown to affect mouse susceptibility to infection. Inbred and congenic mice harboring macrophage-sensitizing Nlrp1b(S/S) alleles (which allow activation of caspase-1 and IL-1β release in response to anthrax lethal toxin challenge) effectively controlled bacterial growth and dissemination when compared to mice having Nlrp1b(R/R) alleles (which cannot activate caspase-1 in response to toxin). Nlrp1b(S)-mediated resistance to infection was not dependent on the route of infection and was observed when bacteria were introduced by either subcutaneous or intravenous routes. Resistance did not occur through alterations in spore germination, as vegetative bacteria were also killed in Nlrp1b(S/S) mice. Resistance to infection required the actions of both caspase-1 and IL-1β as Nlrp1b(S/S) mice deleted of caspase-1 or the IL-1 receptor, or treated with the Il-1 receptor antagonist anakinra, were sensitized to infection. Comparison of circulating neutrophil levels and IL-1β responses in Nlrp1b(S/S),Nlrp1b(R/) (R) and IL-1 receptor knockout mice implicated Nlrp1b and IL-1 signaling in control of neutrophil responses to anthrax infection. Neutrophil depletion experiments verified the importance of this cell type in resistance to B. anthracis infection. These data confirm an inverse relationship between murine macrophage sensitivity to lethal toxin and mouse susceptibility to spore infection, and establish roles for Nlrp1b(S), caspase-1, and IL-1β in countering anthrax infection.     

Anthrax lethal toxin increases superoxide production in murine neutrophils via differential effects on MAPK signaling pathways

The combination of lethal factor and its receptor-binding partner, protective Ag, is termed lethal toxin (LT) and has critical pathogenic activity during infection with Bacillus anthracis. We herein report that anthrax LT binds and enters murine neutrophils, leading to the cleavage of mitogen-activated protein kinase kinase/MEK/MAPKK 1-4 and 6, but not mitogen-activated protein kinase kinase 5 and 7. Anthrax LT treatment of neutrophils disrupts signaling to downstream MAPK targets in response to TLR stimulation. Following anthrax LT treatment, ERK family and p38 phosphorylation are nearly completely blocked, but signaling to JNK family members persists in vitro and ex vivo. In contrast to previous reports involving human neutrophils, anthrax LT treatment of murine neutrophils increases their production of superoxide in response to PMA or TLR stimulation in vitro or ex vivo. Although this enhanced superoxide production correlates with effects due to the LT-induced blockade of ERK signaling, it requires JNK signaling that remains largely intact despite the activity of anthrax LT. These findings reveal a previously unrecognized mechanism through which anthrax LT supports a critical proinflammatory response of murine neutrophils.     

CXCL1/macrophage inflammatory protein-2-induced angiogenesis in vivo is mediated by neutrophil-derived vascular endothelial growth factor-A

The angiogenic activity of CXC-ELR(+) chemokines, including CXCL8/IL-8, CXCL1/macrophage inflammatory protein-2 (MIP-2), and CXCL1/growth-related oncogene-alpha in the Matrigel sponge angiogenesis assay in vivo, is strictly neutrophil dependent, as neutrophil depletion of the animals completely abrogates the angiogenic response. In this study, we demonstrate that mice deficient in the src family kinases, Hck and Fgr (hck(-/-)fgr(-/-)), are unable to develop an angiogenic response to CXCL1/MIP-2, although they respond normally to vascular endothelial growth factor-A (VEGF-A). Histological examination of the CXCL1/MIP-2-containing Matrigel implants isolated from wild-type or hck(-/-)fgr(-/-) mice showed the presence of an extensive neutrophil infiltrate, excluding a defective neutrophil recruitment into the Matrigel sponges. Accordingly, neutrophils from hck(-/-)fgr(-/-) mice normally migrated and released gelatinase B in response to CXCL1/MIP-2 in vitro, similarly to wild-type neutrophils. However, unlike wild-type neutrophils, those from hck(-/-)fgr(-/-) mice were completely unable to release VEGF-A upon stimulation with CXCL1/MIP-2. Furthermore, neutralizing anti-VEGF-A Abs abrogated the angiogenic response to CXCL1/MIP-2 in wild-type mice and CXCL1/MIP-2 induced angiogenesis in the chick embryo chorioallantoic membrane assay, indicating that neutrophil-derived VEGF-A is a major mediator of CXCL1/MIP-2-induced angiogenesis. Finally, in vitro kinase assays confirmed that CXCL1/MIP-2 activates Hck and Fgr in murine neutrophils. Taken together, these data demonstrate that CXCL1/MIP-2 leads to recruitment of neutrophils that, in turn, release biologically active VEGF-A, resulting in angiogenesis in vivo. Our observations delineate a novel mechanism by which CXCL1/MIP-2 induces neutrophil-dependent angiogenesis in vivo.     

Neutrophil Elastase Alters the Murine Gut Microbiota Resulting in Enhanced Salmonella Colonization

The intestinal microbiota has been found to play a central role in the colonization of Salmonella enterica serovar Typhimurium in the gastrointestinal tract. In this study, we present a novel process through which Salmonella benefit from inflammatory induced changes in the microbiota in order to facilitate disease. We show that Salmonella infection in mice causes recruitment of neutrophils to the gut lumen, resulting in significant changes in the composition of the intestinal microbiota. This occurs through the production of the enzyme elastase by neutrophils. Administration of recombinant neutrophil elastase to infected animals under conditions that do not elicit neutrophil recruitment caused shifts in microbiota composition that favored Salmonella colonization, while inhibition of neutrophil elastase reduced colonization. This study reveals a new relationship between the microbiota and the host during infection.     

Involvement of phosphoinositide 3-kinases in neutrophil activation and the development of acute lung injury.

Activated neutrophils contribute to the development and severity of acute lung injury (ALI). Phosphoinositide 3-kinases (PI3-K) and the downstream serine/threonine kinase Akt/protein kinase B have a central role in modulating neutrophil function, including respiratory burst, chemotaxis, and apoptosis. In the present study, we found that exposure of neutrophils to endotoxin resulted in phosphorylation of Akt, activation of NF-kappaB, and expression of the proinflammatory cytokines IL-1beta and TNF-alpha through PI3-K-dependent pathways. In vivo, endotoxin administration to mice resulted in activation of PI3-K and Akt in neutrophils that accumulated in the lungs. The severity of endotoxemia-induced ALI was significantly diminished in mice lacking the p110gamma catalytic subunit of PI3-K. In PI3-Kgamma(-/-) mice, lung edema, neutrophil recruitment, nuclear translocation of NF-kappaB, and pulmonary levels of IL-1beta and TNF-alpha were significantly lower after endotoxemia as compared with PI3-Kgamma(+/+) controls. Among neutrophils that did accumulate in the lungs of the PI3-Kgamma(-/-) mice after endotoxin administration, activation of NF-kappaB and expression of proinflammatory cytokines was diminished compared with levels present in lung neutrophils from PI3-Kgamma(+/+) mice. These results show that PI3-K, and particularly PI3-Kgamma, occupies a central position in regulating endotoxin-induced neutrophil activation, including that involved in ALI.     

Extracellular ATP stimulates elastase secretion from human neutrophils and increases lung resistance and secretion from normal rat airways after intratracheal instillation.

The effect of ATP on intracellular Ca2+ levels and elastase secretion in isolated normal human peripheral blood neutrophils was investigated as was its in vivo effect on lung resistance and mucous secretion. ATP (10(-5) M) increased [Ca2+]i from 61 +/- 3 to 165 +/- 15 nM in nonactivated neutrophils; elastase secretion was increased by 40% from nonactivated neutrophils but was unaffected in fMLP (10(-5) M) activated cells. Instillation of ATP (10(-5) and 10(-3) M) into the airways of brown Norway rats increased both lung resistance and secretion. These findings suggest that aerosolization of ATP into the cystic fibrosis-affected bronchial tree might be hazardous in terms of enhancement of parenchymal damage, which would result from neutrophil elastase release, and in terms of impaired respiratory lung function.     

Bacillus anthracis toxins inhibit human neutrophil NADPH oxidase activity

Bacillus anthracis, the causative agent of anthrax, is a Gram-positive, spore-forming bacterium. B. anthracis virulence is ascribed mainly to a secreted tripartite AB-type toxin composed of three proteins designated protective Ag (PA), lethal factor, and edema factor. PA assembles with the enzymatic portions of the toxin, the metalloprotease lethal factor, and/or the adenylate cyclase edema factor, to generate lethal toxin (LTx) and edema toxin (ETx), respectively. These toxins enter cells through the interaction of PA with specific cell surface receptors. The anthrax toxins act to suppress innate immune responses and, given the importance of human neutrophils in innate immunity, they are likely relevant targets of the anthrax toxin. We have investigated in detail the effects of B. anthracis toxin on superoxide production by primary human neutrophils. Both LTx and ETx exhibit distinct inhibitory effects on fMLP (and C5a) receptor-mediated superoxide production, but have no effect on PMA nonreceptor-dependent superoxide production. These inhibitory effects cannot be accounted for by induction of neutrophil death, or by changes in stimulatory receptor levels. Analysis of NADPH oxidase regulation using whole cell and cell-free systems suggests that the toxins do not exert direct effects on NADPH oxidase components, but rather act via their respective effects, inhibition of MAPK signaling (LTx), and elevation of intracellular cAMP (ETx), to inhibit upstream signaling components mediating NADPH oxidase assembly and/or activation. Our results demonstrate that anthrax toxins effectively suppress human neutrophil-mediated innate immunity by inhibiting their ability to generate superoxide for bacterial killing.     

Delayed treatment with doxycycline has limited effect on anthrax infection in BLK57/B6 mice

Blk57/B6 mice were infected with LD90 dose of Sterne strain anthrax spores subcutaneously and then treated with doxycycline. Doxycycline at a dose of 1.5mg/kg, by intra-peritoneal injection, protected mice from death when given at the same time as spores. When doxycycline administration was delayed 4h survival is 90%. Delay of 24h increased survival time but had no impact on eventual mortality. When doxycycline was delayed 48h, mortality and time to death were comparable to sham injection. Peritoneal macrophages harvested from Blk57/B6 mice were examined for response to anthrax lethal toxin and are shown to be deficient in their ability to produce TNF-alpha and have increased expression of IL-6 compared to RAW 264.7 murine macrophage cell line. These findings suggest that antibiotic therapy has limited effects following lethal anthrax spore challenge, even when the host is of a phenotype that does not produce TNF-alpha in response to anthrax lethal toxin exposure.     

Deletion mutants of protective antigen that inhibit anthrax toxin both in vitro and in vivo

The anthrax toxin complex is primarily responsible for most of the symptoms of anthrax. This complex is composed of three proteins, anthrax protective antigen, anthrax edema factor, and anthrax lethal factor. The three proteins act in binary combination of protective antigen plus edema factor (edema toxin) and protective antigen plus lethal factor (lethal toxin) that paralyze the host defenses and eventually kill the host. Both edema factor and lethal factor are intracellularly acting proteins that require protective antigen for their delivery into the host cell. In this study, we show that deletion of certain residues of protective antigen results in variants of protective antigen that inhibit the action of anthrax toxin both in vitro and in vivo. These mutants protected mice against both lethal toxin and edema toxin challenge, even when injected at a 1:8 ratio relative to the wild-type protein. Thus, these mutant proteins are promising candidates that may be used to neutralize the action of anthrax toxin.     

Lethal toxin actions and their consequences

After entry of infectious anthrax spores into the body, host-specific signals induce spore germination, outgrowth of vegetative bacilli and the expression of lethal toxin and other virulence factors. Anthrax lethal toxin (LeTx) is a virulence factor responsible for the major pathologies seen during systemic anthrax infections. Injection of sterile LeTx into test animals mimics the shock and sudden death seen during active bacterial infections. Once large levels of LeTx are produced within the body, destruction of bacteria by administration of antibiotics is usually unsuccessful. The LeTx is believed to be secreted into the bloodstream where it circulates freely throughout the body and binds and enters host cells. Once in the cytoplasm, the lethal factor acts as a zinc-metalloprotease disrupting normal homoeostatic functions. Macrophages are a uniquely sensitive cell type that seem to be vital global mediators of toxin-induced pathologies. Removal of macrophages from mice renders them insensitive to LeTx challenge. Low levels of lethal toxin induce macrophage production, in vitro, of the shock-inducing cytokines TNF and Il-1beta. Higher levels of LeTx cause over-production of reactive oxygen intermediates, bursting of macrophages and release of mediators of shock. We believe that agents capable of blocking key steps of the lethal toxin cascade may prove useful in combating anthrax pathologies.     

Intracellular calcium antagonist protects cultured peritoneal macrophages against anthrax lethal toxin-induced cytotoxicity

The lethal toxin ofBacillus anthracis is central to the pathogenesis of anthrax. Using primary cultures of mouse peritoneal macrophages, we have demonstrated that intracellular calcium release inhibitors protect against anthrax lethal toxin-induced cytotoxicity. The cytolytic effect of anthrax lethal toxin was markedly reduced by dantrolene, an inhibitor of calcium release from intracellular calcium stores. Pretreatment of macrophages with cyclosporin A, which has been shown to be a potent inhibitor of calcium release from mitochondria, also protected cells against cytotoxicity. These results indicate that calcium release from intracellular store may be an essential step for the propagation of anthrax lethal toxin-induced cell damage in macrophages. Thus our findings suggest that dantrolene, cyclosporin A, and possibly other drugs affecting intracellular calcium pools might be effectively preventing the toxicity from anthrax lethal toxin. This revised version was published online in July 2006 with corrections to the Cover Date.     

Using MT(-/-) mice to study metallothionein and oxidative stress

Mice with null mutations for metallothionein genes MT-1 and MT-2 were used to study the role that metallothionein plays in protecting cellular targets in vivo from oxidative stress. Wild-type (MT(+/+)) and MT-null (MT(-/-)) mice were treated with either saline or zinc and exposed to two types of oxidative stress: gamma-irradiation or 2-nitropropane. There was no alteration in the antioxidant defense system (superoxide dismutase, catalase, or glutathione peroxidase and glutathione levels) to compensate for the lack of the metallothionein in the MT(-/-) mice. The amount of oxidative damage to liver DNA, lipids, and proteins were similar for the MT(-/-) and MT(+/+) mice even though the levels of metallothionein in the livers of the saline- or zinc-pretreated MT(+/+) mice were 5- to 100-fold greater than found in the MT(-/-) mice. To determine if metallothionein can protect mice from the lethal effects of ionizing radiation, the mean survivals of MT(-/-) and MT(+/+) mice exposed to whole body gamma-irradiation were measured and found to be similar. However, the mean survival increased significantly after zinc pretreatment for both the MT(-/-) and MT(+/+) mice. These results demonstrate that tissue levels of metallothionein do not protect mice in vivo against oxidative stress.     

Immunosuppressive action of the lethal toxin of Bacillus anthracis in mice

In experiments on inbred mice infected with B. anthracis capsular strain 71/12 of Tsenkovsky's second vaccine B. anthracis lethal toxin introduced in mixture with spores has been shown to aggravate anthrax infection in CBA mice susceptible to anthrax, while producing a faint effect on the infectious process in BALB mice with hereditary resistance to anthrax. B. anthracis purified edema toxin has been found to produce a weaker aggravating effect with respect to anthrax infection than the lethal toxin. As revealed in these experiments, the capacity of the lethal toxin to suppress the activity of peritoneal macrophages in vitro is the more pronounced, the more resistant to anthrax are the mice used as the donors of these macrophages. The mechanism of hereditary immunity which may ensure resistance to infection in the presence of immunosuppression is discussed.     

Altered CXCR2 signaling in beta-arrestin-2-deficient mouse models

CXCR2 is a G-protein-coupled receptor (GPCR) that binds the CXC chemokines, CXCL1-3 and CXCL5-8, and induces intracellular signals associated with chemotaxis. Many adaptor proteins are actively involved in the sequestration, internalization, and trafficking of CXCR2 and transduction of agonist-induced intracellular signaling. We have previously shown that adaptor protein beta-arrestin-2 (betaarr2) plays a crucial role in transducing signals mediated through CXCR2. To further investigate the role of betaarr2 on CXCR2-mediated signaling during acute inflammation, zymosan-induced neutrophils were isolated from peritoneal cavities of betaarr2-deficient (betaarr2(-/-)) and their wild-type (betaarr2(+/+)) littermate mice, and neutrophil CXCR2 signaling activities were determined by measurement of Ca(2+) mobilization, receptor internalization, GTPase activity, and superoxide anion production. The results showed that the deletion of betaarr2 resulted in increased Ca(2+) mobilization, superoxide anion production, and GTPase activity in neutrophils, but decreased receptor internalization relative to wild-type mice. Two animal models, the dorsal air pouch model and the excisional wound healing model, were used to further study the in vivo effects of betaarr2 on CXCR2-mediated neutrophil chemotaxis and on cutaneous wound healing. Surprisingly, the recruitment of neutrophils was increased in response to CXCL1 in the air pouch model and in the excisional wound beds of betaarr2(-/-) mice. Wound re-epithelialization was also significantly faster in betaarr2(-/-) mice than in betaarr2(+/+) mice. Taken together, the data indicate that betaarr2 is a negative regulator for CXCR2 in vivo signaling.     

Olfactomedin 4 Inhibits Cathepsin C-Mediated Protease Activities, Thereby Modulating Neutrophil Killing of Staphylococcus aureus and Escherichia coli in Mice

Neutrophils kill bacteria generally through oxidative and nonoxidative mechanisms. Whereas much research has focused on the enzymes essential for neutrophil killing, little is known about the regulatory molecules responsible for such killing. In this study, we investigated the role of olfactomedin 4 (OLFM4), an olfactomedin-related glycoprotein, in neutrophil bactericidal capability and host innate immunity. Neutrophils from OLFM4(-/-) mice have increased intracellular killing of Staphylococcus aureus and Escherichia coli in vitro. The OLFM4(-/-) mice have enhanced in vivo bacterial clearance and are more resistant to sepsis when challenged with S. aureus or E. coli by i.p. injection. OLFM4 was found to interact with cathepsin C, a cysteine protease that plays an important role in bacterial killing and immune regulation. We demonstrated that OLFM4 inhibited cathepsin C activity in vitro and in vivo. The cathepsin C activity in neutrophils from OLFM4(-/-) mice was significantly higher than that in neutrophils from wild-type littermate mice. The activities of three serine proteases (neutrophil elastase, cathepsin G, and proteinase 3), which require cathepsin C activity for processing and maturity, were also significantly higher in OLFM4(-/-) neutrophils. The bacterial killing and clearance capabilities observed in OLFM4(-/-) mice that were enhanced relative to wild-type mice were significantly compromised by the additional loss of cathepsin C in mice with OLFM4 and cathepsin C double deficiency. These results indicate that OLFM4 is an important negative regulator of neutrophil bactericidal activity by restricting cathepsin C activity and its downstream granule-associated serine proteases.     

The neutrophil response to polyvinyl sponge implantation

Neutrophil release and migration in mice were studied over a 24-hr period after the sc implantation of a single polyvinyl sponge. The release of neutrophils from the marrow was evaluated by directly counting the residual neutrophils in the femoral marrow of animals with sponges. Sponge and tissue neutrophil content was determined by extraction and assay of myeloperoxidase (MPO), a marker enzyme for neutrophils. A maximum depletion of 48% of the mature neutrophils in the marrow was observed 5 hr after sponge implantation, in keeping with significant release of neutrophils for migration to the sponge. The released cells were not found in the circulating granulocyte pool, since neutropenia was noted. The accumulation of neutrophils in the sponge increased throughout the 24-hr period, whereas in the tissue adjacent to the sponge maximum accumulation of neutrophils occurred within 7 hr. In fact, neutrophils migrated to at least three sites--the sponge, the skin overlying the sponge, and the skin in which an incision had been made to insert the sponge. The sponge content of neutrophils represented 0.3-33% of the neutrophils migrating to the combined lesion (sponge and skin sites). Therefore, if the neutrophil response to foreign body implantation is to be measured in its entirety, it is necessary to quantify not only the neutrophils within the foreign body but also those in the tissues surrounding it. These studies describe an animal model for neutrophil release and migration to tissues following a standard stimulus. It is proposed that this model may be useful in exploring the factors which influence the release and migration of neutrophils in vivo.