Suresh K. Gupta

LUNG INJURY, INFLAMMATION, AND INFLAMMATORY STIMULI IN RATS EXPOSED TO OZONE

The effects of ozone (O3) on airway epithelia, inflammation, and expression of inflammatory stimuli were investigated to delineate the mechanisms of inflam matory reactions relevant to lung injury. Because the airway responses to O3 develop gradually, this investigation included a time-sequence analysis. Rats exposed for 3 h to 1 ppm O3 were studied at 4-h intervals up to 20 h postexposure. Bronchoalveolar lavage fluid (BAL) was analyzed for albumin as an indicator of increased permeability, polymorphonuclear leukocytes (PMNs) to assess the inflammatory status, macrophage inflammatory protein-2 (MIP-2, an inflammatory chemokine), and cell adhesion molecules for their role in inflammation and PMN functions. The time-related increase in album in was matched by a similar significant increase for PMNs, MIP-2, and intercellular adhesion molecule-1 (ICAM-1). However, no marked change occurred for b-2 integrin (CD-18) and leukotriene B4 (LTB4). The results establish a temporal correlation of epithelial permeability with changes in inflammatory activity and stimuli responsible for PMN recruitment in the lung. The observations of elevated MIP-2 and ICAM-1 levels are consistent with their role in injury and inflammation. An early expression of MIP-2 mRNA in BAL cells, that is, immediately post O3 exposure, and the peak increase in BAL MIP-2 levels 4 h later support the chem otactic role of MIP-2 in PMN recruitm ent at 4- and 12-h time points. The rapid drop in MIP-2 and ICAM-1 levels appears to signal the termination of inflammatory cell recruitment, which is accompanied by an onset of recovery.

The enzymatic activity of phosphoglucose isomerase is not required for its cytokine function

PGI is a housekeeping gene encoding phosphoglucose isomerase (PGI) a glycolytic enzyme that also functions as a cytokine (autocrine motility factor (AMF)/neuroleukin/maturation factor) upon secretion from the cell and binding to its 78 kDa seven‐transmembrane domain receptor (gp78/AMF‐R). PGI contains a CXXC motif, characteristic of redox proteins and possibly evolutionarily related to the CC and CXC motif of the chemokine gene family. Using site‐directed mutagenesis, single‐ and double‐deletion (CXC, CC) mutants were created by deleting amino acids 331 and 332 of human PGI, respectively. The mutant proteins lost their enzymatic activity; however, neither of the deletions augmented the proteins’ binding affinity to the receptor and all maintained cytokine function. The results demonstrate that the enzymatic activity of PGI is not essential for either receptor binding or cytokine function of human PGI.

Alteration of epithelial integrity, alkaline phosphatase activity, and fibronectin expression in lungs of rats exposed to ozone.

The deleterious effects of ozone (O3), an oxidant air pollutant, in the lung are dependent on dose and exposure duration and generally evolve with time postexposure. This study characterized the time sequence of epithelial injury and fibronectin expression in the lungs of rats exposed to O3. Bronchoalveolar lavage (BAL) fluid was analyzed for alkaline phosphatase and total protein as markers of epithelial injury and increased permeability, and fibronectin for its role in inflammation and lung injury. The results revealed a time-related increase in total protein in the BAL fluid following a 3-h exposure of rats to 1 ppm O3. The increased protein concentrations peaked at 12 h and then declined, but remained significantly higher than control at 24 h postexposure. A similar time-related significant increase also occurred for BAL fibronectin and alkaline phosphatase activity. However, the return of alkaline phosphatase levels to baseline prior to a comparable reduction in protein levels suggests repair of injured cells, but a delay in the formation of epithelial junctions that limit the transfer of serum proteins to air spaces. By cytochemistry, alkaline phosphatase activity was detected in association with lung type II epithelial cells and in BAL polymorphonuclear leukocytes (PMNs), but not in macrophages. While a significant increase in cytochemically detectable alkaline phosphatase resulted from the increase in PMN number following O3 exposure, mononuclear cells constituted the primary cell type responsible for fibronectin mRNA upregulation. While the cytochemical observations support the role of inflammatory cells in the injury process, the comparability of temporal changes in BAL protein, fibronectin, and alkaline phosphatase suggests a mechanistic role for fibronectin in lung injury.

Attenuation of ozone-induced lung injury by interleukin-10

Ozone (O3), an oxidant air pollutant, is capable of producing pulmonary inflammation and injury. Exposure to O3 results in the release of inflammatory cytokines including tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) by alveolar macrophages. In addition, O3 exposure results in an increased expression of the inducible isoform of nitric oxide synthetase (iNOS). Interleukin-10 (IL-10) is an anti-inflammatory cytokine which inhibits the synthesis of TNF-alpha and IL-1 by macrophages and decreases the expression of iNOS. To test the protective properties of IL-10 in vivo, on the pulmonary injury induced by O3 exposure, we intratracheally instilled rat recombinant IL-10 1 h prior to O3 exposure (0.8 ppm x 3 h). Approximately 10-12 h following exposure, the animals were sacrificed and the bronchoalveolar lavage fluid (BALF) collected. The quantification of albumin, protein and fibronectin in the BALF provided a means of assessing pulmonary injury while the analysis of the BALF cells reflected the inflammatory response. Ozone exposure resulted in a significant (P<0.05) increase in BALF albumin, protein and fibronectin content as compared to air-exposed controls. In addition, significant increases in the percentage of BALF polymorphonuclear leukocytes (PMNs) and tissue expression of fibronectin mRNA were observed. The intratracheal instillation of IL-10 prior to O3 exposure resulted in a significant reduction in BALF albumin, protein and fibronectin content, and lung fibronectin mRNA as compared to O3 exposure alone. The data shows that IL-10, when given intratracheally, significantly reduces the pulmonary injury following O3 exposure in the rat. However, since the PMNs and the levels of albumin, protein and fibronectin in the IL-10 treated group did not reach baseline values, we conclude that other mediators of inflammation and injury not regulated by IL-10 also contribute to the pathophysiology of O3-induced lung injury.

Characterization of P. aeruginosa pili binding human corneal epithelial proteins.

Soluble human corneal epithelial proteins (hcep) to which P. aeruginosa pili bind were identified and characterized using gel electrophoresis and Western blotting techniques. Pilus binding proteins were identified using nitrocellulose membrane blots of one dimensional polyacrylamide gels (1-D-SDS-PAGE) of solubilized hcep and a pilus overlay assay. Five major proteins of approximate molecular weights < 21, 38, 45, 66, 97 (a doublet) kilodaltons (kDa) and two additional proteins of > 97 kDa bound pili using an overlay assay and immunoblotting with the monoclonal antibody (MAb) XLR-3, specific for pili. Several of these pili binding proteins were confirmed as single proteins using a similar pilus overlay assay and blotted proteins from two-dimensional polyacrylamide gels (2-D-SDS-PAGE) of hcep. A solid-phase binding assay confirmed that pili binding to hcep was specific, competitive and saturable. The importance of the carbohydrate portions of corneal pili binding proteins was assessed by preincubation of 1-D gel blots of hcep or dot blots of selected eluted pilus binding proteins ( < 21, 38, 45, 66 and 97 kDa) with periodic acid. Mild periodate oxidation of blots before pilus overlay assay completely abolished pili binding. The role of glycosylation of proteins also was assessed using 1-D blots of hcep or dot blots of eluted pilus binding proteins. These blots were preincubated with different lectins before incubation with pili in the pilus overlay assay. Of several lectins examined, only sConA, which recognizes terminal mannose residues, prevented pili binding in the pilus overlay assay. These studies provide evidence that several human corneal epithelial glycosylated proteins provide receptor sites for bacterial pili binding, and that the binding of pili to these proteins is specific, competitive and saturable. They also show that the carbohydrate mannose functions as an integral component of hcep pili binding receptors.

Anti-receptor antibodies inhibit Pseudomonas aeruginosa binding to the cornea and prevent corneal perforation

A polyclonal antibody (pAb) against gangliotetraosylceramide (asialo GM1), a glycolipid to which bacterial pili and LPS bind, and a mAb against a 66 kDa pilus‐binding protein purified from adult mouse corneal epithelium were used to determine if antibodies against host receptors for bacterial adhesins could inhibit bacterial binding to wounded corneal epithelium and protect ocularly challenged mice from corneal perforation when topically applied. Bacteria were mixed with anti‐66 kDa mAb, a mixture of anti asialo GM1 pAb and anti‐66 kDa mAb, an irrelevant control mAb (anti‐human histocompatibility Ag HLA‐DR5) or PBS prior to application to scarified corneas in organ culture. The combination of the two antibodies or the anti‐66 kDa mAb alone was effective in reducing bacterial adherence compared with either PBS or the antibody control. To determine if these antibodies were protective in vivo, corneas of C57BL/6J mice were scarified and inoculated with Pseudomonas aeruginosa. Eyes were treated topically with anti‐asialo GM1 pAb, anti‐66 kDa mAb, a mixture of the two or control mouse serum. More serum‐treated corneas perforated compared to corneas from any other group (p≤= 0.005) by 30 days post infection. Treatment with a combination of the two antibodies resulted in significantly less corneal pathology 30 days p.i. when compared to any other treatment (p≤= 0.005). These data provide evidence that antibodies against host corneal receptors significantly inhibit bacterial binding in vitro and when applied topically in vivo, lessen the severity of ocular disease characteristic of P. aeruginosa keratitis.

Enhancement of fibronectin expression in rat lung by ozone and an inflammatory stimulus

This study investigated the relationship of fibronectin expression and induction of pulmonary inflammation by ozone (O3). Rats were exposed to 0.8 parts/million O3 to induce lung inflammation. A second inflammatory stimulus, rabbit serum, was applied intratracheally to augment O3-induced inflammation. Bronchoalveolar lavage fluid (BALF) and lung tissues were analyzed for fibronectin protein and mRNA expression. Blood plasma was analyzed to investigate the potential of a minimally invasive procedure in predicting lung inflammation and fibronectin levels. Significant increases in the levels of fibronectin protein in the BALF and lung tissue after O3 exposure were further enhanced by pretreatment with normal serum. An increase in fibronectin mRNA following O3 exposure was also enhanced by serum pretreatment, which by itself had no effect on lung fibronectin mRNA expression. Plasma fibronectin levels were comparable in air-PBS and O3-PBS groups but increased in the O3-serum group. The results suggest leakage of fibronectin from blood plasma into the lung following intratracheal application of rabbit serum and upregulation of local synthesis following O3 exposure.