Cigarette smoke-induced lung emphysema in mice is associated with prolyl endopeptidase, an enzyme involved in collagen breakdown

There is increasing evidence that the neutrophil chemoattractant proline-glycine-proline (PGP), derived from the breakdown of the extracellular matrix, plays an important role in neutrophil recruitment to the lung. PGP formation is a multistep process involving neutrophils, metalloproteinases (MMPs), and prolyl endopeptidase (PE). This cascade of events is now investigated in the development of lung emphysema. A/J mice were whole body exposed to cigarette smoke for 20 wk. After 20 wk or 8 wk after smoking cessation, animals were killed, and bronchoalveolar lavage fluid and lung tissue were collected to analyze the neutrophilic airway inflammation, the MMP-8 and MMP-9 levels, the PE activity, and the PGP levels. Lung tissue degradation was assessed by measuring the mean linear intercept. Additionally, we investigated the effect of the peptide L-arginine-threonine-arginine (RTR), which binds to PGP sequences, on the smoke-induced neutrophil influx in the lung after 5 days of smoke exposure. Neutrophilic airway inflammation was induced by cigarette smoke exposure. MMP-8 and MMP-9 levels, PE activity, and PGP levels were elevated in the lungs of cigarette smoke-exposed mice. PE was highly expressed in epithelial and inflammatory cells (macrophages and neutrophils) in lung tissue of cigarette smoke-exposed mice. After smoking cessation, the neutrophil influx, the MMP-8 and MMP-9 levels, the PE activity, and the PGP levels were decreased or reduced to normal levels. Moreover, RTR inhibited the smoke-induced neutrophil influx in the lung after 5 days' smoke exposure. In the present murine model of cigarette smoke-induced lung emphysema, it is demonstrated for the first time that all relevant components (neutrophils, MMP-8, MMP-9, PE) involved in PGP formation from collagen are upregulated in the airways. Together with MMPs, PE may play an important role in the formation of PGP and thus in the pathophysiology of lung emphysema.     

The neutrophil migration induced by tumour necrosis factor alpha in mice is unaffected by glucocorticoids

Macrophages harvested from the peritoneal cavities of rats release a neutrophil chemotactic factor (MNCF) in response to stimulation with Gram-negative bacterial lipopolysaccharide (LPS). MNCF has been shown to be active in rats treated with dexamethasone, a glucocorticoid that usually inhibits the neutrophil migration induced in this species by interleukin (IL)-1, tumour necrosis factor alpha (TNFalpha), IL-8, C5a and leukotriene B(4) (LTB(4)). Here we report that macrophages harvested from peritoneal cavities of mice, and stimulated in vitro with LPS, also release a factor that induces neutrophil migration in dexamethasone-treated animals. This chemotactic activity was neutralized by the incubation of the LPS-stimulated macrophage supernatants with a purified polyclonal IgG anti-mouse TNFalpha. In addition, significant amounts of TNF were detected in the supernatants. The neutrophil migration induced by intraperitoneal administration of recombinant murine TNFalpha was also unaffected by pretreatment of the mice with dexamethasone. Moreover, neutrophil migration induced by intraperitoneal injection of LPS was completely blocked by pretreatment of the mice with a monoclonal antibody against murine TNFalpha. In conclusion, our results support the hypothesis that, in contrast to the role of TNF in rats (where it indirectly induces neutrophil migration), in mice, it may be an important mediator in the recruitment of neutrophils to inflammatory sites.     

Cell adhesion molecules involved in the leukocyte recruitment induced by venom of the snake Bothrops jararaca

It has been shown that Bothrops jararaca venom (BjV) induces a significant leukocyte accumulation, mainly neutrophils, at the local of tissue damage. Therefore, the role of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1), LECAM-1, CD18, leukocyte function-associated antigen-1 (LFA-1) and platelet endothelial cell adhesion molecule-1 (PECAM-1) on the BjV-induced neutrophil accumulation and the correlation with release of LTB4, TXA2, tumor necrosis factor-alpha, interleukin (IL)-1 and IL-6 have been investigated. Anti-mouse LECAM-1, LFA-1, ICAM-1 and PECAM-1 monoclonal antibody injection resulted in a reduction of 42%, 80%, 66% and 67%, respectively, of neutrophil accumulation induced by BjV (250 microg/kg, intraperitoneal) injection in male mice compared with isotype-matched control injected animals. The anti-mouse CD18 monoclonal antibody had no significant effect on venom-induced neutrophil accumulation. Concentrations of LTB(4), TXA(2), IL-6 and TNF-alpha were significant increased in the peritoneal exudates of animals injected with venom, whereas no increment in IL-1 was detected. This results suggest that ICAM-1, LECAM-1, LFA-1 and PECAM-1, but not CD18, adhesion molecules are involved in the recruitment of neutrophils into the inflammatory site induced by BjV. This is the first in vivo evidence that snake venom is able to up-regulate the expression of adhesion molecules by both leukocytes and endothelial cells. This venom effect may be indirect, probably through the release of the inflammatory mediators evidenced in the present study.     

A physiological function for apolipoprotein(a): a natural regulator of the inflammatory response

Structural similarities between apolipoprotein(a) (apo(a)), the unique apoprotein of lipoprotein(a), and plasminogen, the zymogen of plasmin, can interfere with functions of plasmin (ogen) in vitro. The purpose of this study was to evaluate the role of apo(a) in inflammation in vivo using apo(a) transgenic mice and to determine if effects are plasminogen-dependent using backgrounds that are either plasminogen-replete or plasminogen-deficient. After administration of peritoneal inflammatory stimuli, thioglycollate, bioimplants or lipopolysaccharide, the number of responding peritoneal neutrophils and macrophages were quantified. Apo(a), in either wild-type or plasminogen deficient backgrounds, inhibited neutrophil recruitment but had no effect on plasminogen-dependent macrophage recruitment. Macrophage-inflammatory protein-2, a neutrophil chemokine, was reduced in apo(a) mice, and injection of this chemokine prior to thioglycollate restored neutrophil recruitment in apo(a) transgenic mice. In the lipopolysaccharide model, mice with apo(a), unlike mice without apo(a), did not increase neutrophil recruitment in response to the stimulus. In the bioimplant model, neutrophil recruitment and neutrophil cytokines were reduced in apo(a)tg mice but only in a plasminogen-deficient background. These results indicate for the first time that apo(a), independent of plasminogen interaction, inhibits neutrophil recruitment in vivo in diverse peritoneal inflammatory models. Hence, apo(a) may function as a cell specific suppressor of the inflammatory response.     

ICAM-1 and LFA-1 play critical roles in LPS-induced neutrophil recruitment into the alveolar space

Neutrophil recruitment into lung constitutes a major response to airborne endotoxins. In many tissues endothelial intercellular adhesion molecule-1 (ICAM-1) interacts with lymphocyte function associated antigen-1 (LFA-1) on neutrophils, and this interaction plays a critical role in neutrophil recruitment. There are conflicting reports about the role of ICAM-1 in neutrophil recruitment into lungs. We studied neutrophil recruitment into alveolar space in a murine model of aerosolized LPS-induced lung inflammation. LPS induces at least a 100-fold increase in neutrophil numbers in alveolar space, as determined by flow cytometry of bronchoalveolar lavage fluid. Neutrophil recruitment was reduced by 54% in ICAM-1 null mice and by 45% in LFA-1 null mice. In wild-type mice treated with anti-ICAM-1 and anti-LFA-1 antibodies, there was 51 and 58% reduction in the neutrophil recruitment, respectively. In chimeric mice, generated by the transplantation of mixtures of bone marrows from LFA-1 null and wild-type mice, the normalized recruitment of LFA-1 null neutrophils was reduced by 60% compared with wild-type neutrophils. Neither the treatment of ICAM-1 null mice with a function-blocking antibody to LFA-1 nor the treatment of LFA-1 null mice with anti-ICAM-1 antibody resulted in further reduction in the recruitment compared with untreated ICAM-1 null and LFA-1 null mice. We conclude that ICAM-1 and LFA-1 play critical roles in the recruitment of neutrophils into the alveolar space in aerosolized LPS-induced lung inflammation, and LFA-1 serves as a ligand of ICAM-1 in the lung.     

Role of multidrug resistance P-glycoprotein in the secretion of aldosterone by human adrenal NCI-H295 cells

We determinedthe role of the multidrug resistance (MDR1) gene product,P-glycoprotein (PGP), in the secretion of aldosterone by the adrenalcell line NCI-H295. Aldosterone secretion is significantly decreased bythe PGP inhibitors verapamil, cyclosporin A (CSA), PSC-833, andvinblastine. Aldosterone inhibits the efflux of the PGP substraterhodamine 123 from NCI-H295 cells and from human mesangial cells(expressing PGP). CSA, verapamil, and the monoclonal antibody UIC2significantly decreased the efflux of fluorescein-labeled (FL)-aldosterone microinjected into NCI-H295 cells. In MCF-7/VP cells,expressing multidrug resistance-associated protein (MRP) but not PGP,and in the parental cell line MCF7 (expressing no MRP andno PGP), the efflux of microinjected FL-aldosterone was slow. In BC19/3cells (MCF7 cells transfected with MDR1), the efflux of FL-aldosteronewas rapid and it was inhibited by verapamil, indicating thattransfection with MDR1 cDNA confers the ability to transportFL-aldosterone. These results strongly indicate that PGP plays a rolein the secretion of aldosterone by NCI-H295 cells and in other cellsexpressing MDR1, including normal adrenal cells.

     

Inflammasome-mediated production of IL-1beta is required for neutrophil recruitment against Staphylococcus aureus in vivo

IL-1R activation is required for neutrophil recruitment in an effective innate immune response against Staphylococcus aureus infection. In this study, we investigated the mechanism of IL-1R activation in vivo in a model of S. aureus infection. In response to a S. aureus cutaneous challenge, mice deficient in IL-1beta, IL-1alpha/IL-1beta, but not IL-1alpha, developed larger lesions with higher bacterial counts and had decreased neutrophil recruitment compared with wild-type mice. Neutrophil recruitment and bacterial clearance required IL-1beta expression by bone marrow (BM)-derived cells and not by non-BM-derived resident cells. In addition, mice deficient in the inflammasome component apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) had the same defects in neutrophil recruitment and host defense as IL-1beta-deficient mice, demonstrating an essential role for the inflammasome in mediating the production of active IL-1beta to promote neutrophil recruitment in host defense against S. aureus. This finding was further supported by the ability of recombinant active IL-1beta to control the infection and promote bacterial clearance in IL-1beta-deficient mice. These studies define a key host defense circuit where inflammasome-mediated IL-1beta production by BM-derived cells signals IL-1R on non-BM-derived resident cells to activate neutrophil recruitment in the innate immune response against S. aureus in vivo.     

Pulmonary inflammation induced by high-stretch ventilation is mediated by tumor necrosis factor signaling in mice

Although high-stretch mechanical ventilation has been demonstrated to induce lung inflammation, the roles of soluble mediators, in particular TNF, remain controversial. We have previously shown in mice that high-stretch ventilation, in the absence of preceding lung injury, induces expression of bioactive TNF in lung lavage fluid early in the course of injury, but the biological significance of this, if any, has yet to be determined. We therefore investigated the pulmonary inflammatory response to a transient period of high-stretch ventilation in anesthetized mice lacking TNF receptors and mice treated with anti-TNF antibodies. A standardized stretch-induced lung injury (assessed by lung mechanics, blood gases, and lavage protein content), followed by noninjurious low-stretch ventilation for 3 h, produced significant alveolar neutrophil infiltration in wild-type mice. However, neutrophil recruitment was substantially attenuated in TNF receptor double knockout mice and in wild-type mice treated with intratracheal anti-TNF antibody. This attenuation was not associated with decreased concentrations of neutrophil attractant CXC chemokines (macrophage inflammatory protein-2 and keratinocyte-derived chemokine) in lavage fluid. In contrast to intratracheal antibody, intravenous anti-TNF antibody did not reduce neutrophil infiltration, suggesting that the role of TNF signaling is localized within the alveolar space and does not require decompartmentalization of TNF into the circulation. These findings provide the first direct evidence that pulmonary inflammation induced by high-stretch ventilation without underlying lung injury possesses a significant TNF-dependent component. The results suggest a potential for regional anti-TNF treatment in attenuating stretch-induced pulmonary inflammation.     

Nucleotide-binding oligomerization domain 1 mediates recognition of Clostridium difficile and induces neutrophil recruitment and protection against the pathogen

Clostridium difficile is a Gram-positive obligate anaerobic pathogen that causes pseudomembranous colitis in antibiotics-treated individuals. However, host immune protective mechanisms against C. difficile are largely unknown. In this study, we show that C. difficile possesses potent stimulatory activity for nucleotide-binding oligomerization domain 1 (Nod1), an intracellular pattern recognition molecule that senses bacterial peptidoglycan-related molecules. Nod1(-/-), but not Nod2(-/-), mice exhibited increased lethality in response to C. difficile intestinal infection despite comparable levels of intestinal damage and epithelial permeability in Nod1(-/-) and control mice. The enhanced lethality was accompanied by impaired C. difficile clearance, increased bacterial translocation, and elevated levels of endotoxin and IL-1β in the serum of Nod1(-/-) mice. Histological and flow cytometric analyses revealed that Nod1(-/-) mice had defective recruitment of neutrophils, but not macrophages, to the intestine after C. difficile infection. The reduced recruitment of neutrophils correlated with impaired production of CXCL1, but not CCL2, XCL1, and other cytokines/chemokines, in infected Nod1(-/-) mice. The influx of neutrophils also was reduced when C. difficile was administered i.p., suggesting that Nod1 directly recognizes C. difficile to induce the recruitment of neutrophils to the infected site. These results indicate that Nod1 regulates host susceptibility to C. difficile and suggest that Nod1-mediated neutrophil recruitment is an important immune response against the enteric pathogen.     

Tumor Necrosis Factor,but Not Neutrophils,Alters the Metabolic Profile in Acute Experimental Arthritis

Metabolic alterations are associated with arthritis apart from obesity. However, it is still unclear which is the underlying process behind these metabolic changes. Here, we investigate the role of tumor necrosis factor (TNF) in this process in an acute model of antigen-induced arthritis (AIA). Immunized male BALB/c mice received an intra-articular injection of PBS (control) or methylated bovine serum albumin (mBSA) into their knees, and were also pre-treated with different drugs: Etanercept, an anti-TNF drug, DF2156A, a CXCR1/2 receptor antagonist, or a monoclonal antibody RB6-8C5 to deplete neutrophils. Local challenge with mBSA evoked an acute neutrophil influx into the knee joint, and enhanced the joint nociception, along with a transient systemic metabolic alteration (higher levels of glucose and lipids, and altered adipocytokines). Pre-treatment with the conventional biological Etanercept, an inhibitor of TNF action, ameliorated the nociception and the acute joint inflammation dominated by neutrophils, and markedly improved many of the altered systemic metabolites (glucose and lipids), adipocytokines and PTX3. However, the lessening of metabolic changes was not due to diminished accumulation of neutrophils in the joint by Etanercept. Reduction of neutrophil recruitment by pre-treating AIA mice with DF2156A, or even the depletion of these cells by using RB6-8C5 reduced all of the inflammatory parameters and hypernociception developed after AIA challenge, but could not prevent the metabolic changes. Therefore, the induction of joint inflammation provoked acute metabolic alterations which were involved with TNF. We suggest that the role of TNF in arthritis-associated metabolic changes is not due to local neutrophils, which are the major cells present in this model, but rather due to cytokines.     

Endogenous nitric oxide inhibits neutrophil adherence to lung epithelial cells to modulate interleukin-8 release

To investigate the effect of neutrophil adherence to epithelial cells on the release of interleukin 8 (IL-8), we measured neutrophil adherence in the presence or absence of IFN-gamma+TNF-alpha+IL-1beta (cytomix) stimulation on cultured A549 epithelial cells. The extent of neutrophil adherence to A549 epithelial cells was measured and the concomitant production of IL-8 and nitrite were assayed. The roles of adhesion molecules and nitrite in modulation of neutrophil adherence were examined by pretreatment with oversaturating ICAM-1 blocking antibody and L-NAME (1 mM), respectively. There was a time-dependent spontaneous and cytomix-induced release of IL-8 from epithelial cells, as well as a time-dependent increase in the magnitude of neutrophil adherence to epithelial cells. Stimulation of epithelial cells with cytomix induced a further increase in neutrophil adherence. Pretreatment with oversaturated ICAM-1 monoclonal antibody inhibited neutrophil adherence with or without cytomix stimulation. The inhibition of neutrophil adherence to epithelial cells with ICAM-1 monoclonal antibody or a semipermeable membrane downregulated the release of IL-8 with or without cytomix stimulation. Stimulation with cytomix decreased nitrite production. Both neutrophil adherence and L-NAME pretreatment significantly inhibited the production of nitrite. The inhibition of neutrophil adherence to epithelial cells with ICAM-1 monoclonal antibody or a semipermeable membrane upregulated nitrite production. Pretreatment with L-NAME failed to modify the spontaneous release of IL-8, but significantly enhanced the response to adherence and cytomix. In conclusion, endogenous nitric oxide may play a role in preventing neutrophil adherence to lung epithelial cells, thus modulating concomitant IL-8 release.     

Inhibition of high-mobility group box 1 protein (HMGB1) enhances bacterial clearance and protects against Pseudomonas Aeruginosa pneumonia in cystic fibrosis

Pulmonary infection with Pseudomonas aeruginosa and neutrophilic lung inflammation significantly contribute to morbidity and mortality in cystic fibrosis (CF). High-mobility group box 1 protein (HMGB1), a ubiquitous DNA binding protein that promotes inflammatory tissue injury, is significantly elevated in CF sputum. However, its mechanistic and potential therapeutic implications in CF were previously unknown. We found that HMGB1 levels were significantly elevated in bronchoalveolar lavage fluids (BALs) of CF patients and cystic fibrosis transmembrane conductance regulator (CFTR )(-/-) mice. Neutralizing anti-HMGB1 monoclonal antibody (mAb) conferred significant protection against P. aeruginosa-induced neutrophil recruitment, lung injury and bacterial infection in both CFTR(-/-) and wild-type mice. Alveolar macrophages isolated from mice treated with anti-HMGB1 mAb had improved phagocytic activity, which was suppressed by direct exposure to HMGB1. In addition, BAL from CF patients significantly impaired macrophage phagocytotic function, and this impairment was attenuated by HMGB1-neutralizing antibodies. The HMGB1-mediated suppression of bacterial phagocytosis was attenuated in macrophages lacking toll-like receptor (TLR)-4, suggesting a critical role for TLR4 in signaling HMGB1-mediated macrophage dysfunction. These studies demonstrate that the elevated levels of HMGB1 in CF airways are critical for neutrophil recruitment and persistent presence of P. aeruginosa in the lung. Thus, HMGB1 may provide a therapeutic target for reducing bacterial infection and lung inflammation in CF.     

The Role of Heparanase in Pulmonary Cell Recruitment in Response to an Allergic but Not Non-Allergic Stimulus

Heparanase is an endo-β-glucuronidase that specifically cleaves heparan sulfate proteoglycans in the extracellular matrix. Expression of this enzyme is increased in several pathological conditions including inflammation. We have investigated the role of heparanase in pulmonary inflammation in the context of allergic and non-allergic pulmonary cell recruitment using heparanase knockout (Hpa^-/-) mice as a model. Following local delivery of LPS or zymosan, no significant difference was found in the recruitment of neutrophils to the lung between Hpa^-/- and wild type (WT) control. Similarly neutrophil recruitment was not inhibited in WT mice treated with a heparanase inhibitor. However, in allergic inflammatory models, Hpa^-/- mice displayed a significantly reduced eosinophil (but not neutrophil) recruitment to the airways and this was also associated with a reduction in allergen-induced bronchial hyperresponsiveness, indicating that heparanase expression is associated with allergic reactions. This was further demonstrated by pharmacological treatment with a heparanase inhibitor in the WT allergic mice. Examination of lung specimens from patients with different severity of chronic obstructive pulmonary disease (COPD) found increased heparanase expression. Thus, it is established that heparanase contributes to allergen-induced eosinophil recruitment to the lung and could provide a novel therapeutic target for the development of anti-inflammatory drugs for the treatment of asthma and other allergic diseases.     

A novel proteolytic cascade generates an extracellular matrix-derived chemoattractant in chronic neutrophilic inflammation

Chronic neutrophilic inflammation is a manifestation of a variety of lung diseases including cystic fibrosis (CF). There is increasing evidence that fragments of extracellular matrix proteins, such as collagen and elastin, play an important role in inflammatory cell recruitment to the lung in animal models of airway inflammation. Unfortunately, the association of these peptides with human disease and the identification of therapeutic targets directed toward these inflammatory pathways have remained elusive. In this study, we demonstrate that a novel extracellular matrix-derived neutrophil chemoattractant, proline-glycine-proline (PGP), acts through CXC receptors 1 and 2 on neutrophils, similar to N-acetylated proline-glycine-proline (N-alpha-PGP). We describe the specific multistep proteolytic pathway involved in PGP generation from collagen, involving matrix metalloproteases 8 and 9 and prolyl endopeptidase, a serine protease for which we identify a novel role in inflammation. PGP generation correlates closely with airway neutrophil counts after administration of proteases in vivo. Using CF as a model, we show that CF sputum has elevated levels of PGP peptides and that PGP levels decline during the course of CF inpatient therapy for acute pulmonary exacerbation, pointing to its role as a novel biomarker for this disease. Finally, we demonstrate that CF secretions are capable of generating PGP from collagen ex vivo and that this generation is significantly attenuated by the use of inhibitors directed toward matrix metalloprotease 8, matrix metalloprotease 9, or prolyl endopeptidase. These experiments highlight unique protease interactions with structural proteins regulating innate immunity and support a role for these peptides as novel biomarkers and therapeutic targets for chronic, neutrophilic lung diseases.     

The selective inhibition of inducible nitric oxide synthase prevents intestinal ischemia-reperfusion injury in mice.

Nitric oxide (NO) involvement in intestinal ischemia-reperfusion (I/R) injury has been widely suggested but its protective or detrimental role remains still question of debate. Here, we examine the impact of supplementation or inhibition of NO availability on intestinal dysmotility and inflammation caused by mesenteric I/R in mice. Ischemia 45min and reperfusion 24h were performed by superior mesenteric artery occlusion in female Swiss mice. Saline-treated sham-operated (S) or normal mice without surgery (N) served as controls. Drugs were subcutaneously injected 0, 4, 8, and 18 h after ischemia. Upper gastrointestinal transit (GIT, estimated through black marker gavage), intestinal myeloperoxidase activity (MPO), intestinal malondialdehyde levels (MDA), Evans blue extravasation (EB), intestinal histological damage, and mean arterial pressure (MAP) were considered. In I/R mice, GIT was significantly delayed compared to S and N groups; MPO activity and EB extravasation enhanced, whereas MDA levels did not change. Compared to N and S groups, in I/R mice selective iNOS inhibitor P-BIT significantly prevented motor, MPO and EB changes; putative iNOS inhibitor aminoguanidine significantly counteracted GIT delay but not neutrophil recruitment and the increase in vascular permeability; NOS inhibitor l-NAME and NO precursor l-arginine were scarcely or no effective. Furthermore, in S mice aminoguanidine caused a significant increase of MPO activity reverted by H(1) histamine receptor antagonist pre-treatment. Unlike P-BIT, aminoguanidine and l-NAME injection increased MAP. These findings confirm a detrimental role for iNOS-derived NO overproduction during reperfusion. Aminoguanidine-associated neutrophil recruitment suggests that this drug could act through mechanisms additional to iNOS inhibition involving both eNOS blockade, as indicated by its hemodynamic effects, and indirect activation of H(1) histamine receptors.     

Expression of the beta6 integrin subunit is associated with sites of neutrophil influx in lung epithelium.

Inhalation of ozone by Rhesus monkeys results in epithelial injury and granulocyte influx in both conducting airways and respiratory bronchioles. We have reported that ozone-induced neutrophil recruitment and subsequent epithelial repair can be inhibited in vivo with a CD18 antibody. The antibody-mediated effect is abrogated by local instillation of C5a (a CD18-independent neutrophil chemoattractant), thereby demonstrating a role for neutrophils in lung epithelial repair processes. As an extension of this study, we examined the effect of ozone and neutrophil influx on epithelial expression of the beta6 integrin, an adhesion molecule associated with proliferation and repair. Expression of beta6 integrin was determined by immunohistochemistry for ozone-exposed monkeys treated with either control immunoglobulins or a CD18 antibody. The tracheal epithelium of ozone-exposed monkeys treated with control immunglobulins expressed the beta6 integrin. In contrast, the tracheal epithelium of ozone-exposed monkeys treated with CD18 antibody exhibited very low to undetectable expression of beta6 integrin. In association with C5a instillation and neutrophil influx, beta6 integrin was also observed in respiratory bronchiolar epithelium from both control and ozone-exposed animals. These findings cumulatively suggest that lung epithelial cell expression of beta6 integrin is associated with sites of neutrophil recruitment.     

Detection of minor immunological differences among human "universal-type" alkaline phosphatases

Two clones of monoclonal antibodies against swine alkaline phosphatase (ALPase; orthophosphoric monoester phosphohydrolase, alkaline optimum, EC 3.1.3.1), which were useful in distinguishing human kidney and bone ALPases from liver ALPase, were successfully raised in mice. On the other hand, polyclonal antibody cross-reacted not only with human kidney ALPase but also with all other human universal type ALPases. The difference in cross-reactivity of monoclonal and polyclonal antibodies may be caused by the specific antigenicity of human enzymes. The monoclonal antibodies were able to recognize minor heterogeneity that could not be distinguished by their enzymatic properties. The present monoclonal antibody preparations will be utilized for clinical as well as basic investigations to detect minor heterogeneity among universal-type ALPases.     

Characterization of a Monoclonal Antibody Specific for Lipoteichoic Acid from Various Gram-Positive Bacteria

A hybrid cell line, 3G6, producing monoclonal antibody (mAb) against the polyglycerophosphate (PGP) backbone of lipoteichoic acids has been derived by the polyethylene glycol-induced fusion of mouse myeloma cells and spleen cells from mice immunized with partially purified glucosyltransferase from culture supernatant of Streptococcus mutans strain 6715. Immunodiffusion tests and ELISA revealed that the antibody reacted with purified PGP from group A Streptococcus pyogenes strain Sv as well as crude phenol-water and saline extracts of various gram-positive bacteria except for a few species such as biotype B S. sanguis, Micrococcus sp., and Actinomyces viscosus. Whole cells of serotype b S. mutans and Staphylococcus epidermidis were agglutinated upon addition of 3G6 mAb, while those of most other species were not significantly affected by this procedure. A hapten inhibition study showed that glycerophosphate was only a potent inhibitor of passive hemagglutination reactions between LTA coated sheep erythrocytes and 3G6 mAb.