Immunogenicity, safety and lot consistency in adults of a chromatographically purified Vero-cell rabies vaccine: a randomized, double-blind trial with human diploid cell rabies vaccine.

The immunogenicity and safety of a chromatographically purified rabies vaccine (CPRV) was evaluated using US veterinary medical students. In the first study, 242 healthy adults were enrolled in a randomized, modified double-blind, multicenter trial and received five doses of either CPRV or human diploid cell vaccine (HDCV) by intramuscular injection on days 0, 3, 7, 14, and 28 concurrently with human rabies immunoglobulin in a simulated post-exposure prophylaxis regimen. Post-immunization titers in the CPRV and HDCV groups reached 0.5 IU/ml (the WHO-recommended minimally acceptable titer) or greater in all subjects in both vaccine groups by day 14 and remained above that level through day 90. In the second study, 438 healthy adults were enrolled in a randomized, double-blind, multicenter trial and assigned to receive five doses from one of three lots of CPRV by intramuscular injection on days 0, 3, 7, 14, and 28 in a simulated post-exposure prophylaxis regimen to evaluate lot consistency. Post-immunization titers rapidly increased to over 0.5 IU/ml by day 14 for all subjects and remained above that level through day 42 when the study was terminated. The three lots were considered equivalent. The percentage of subjects with at least one local reaction during the five-dose regimen was slightly lower in the CPRV group than in the HDCV group (P=0.06). The most frequently reported local reaction for all doses of vaccine was pain at the injection site. Headache, myalgia, and malaise were the most frequently reported systemic events. The percentage of subjects with at least one systemic event was significantly lower for CPRV (P=0.0084). No vaccine-related serious adverse reaction was reported in these studies. The results of these studies indicate that CPRV administered intramuscularly to healthy adults is immunogenic and is associated with fewer local and systemic reactions than HDCV.

Pattern of cytokine and adhesion molecule mRNA in hapten-induced relapsing colon inflammation in the rat.

We examined the mRNA expression of cytokines, chemokines, integrins, and selectins in colon lesions of rat colitis with a semi-quantitative RT-PCR assay. Rat colitis was induced by trinitrobenzene sulfonic acid (TNBS) in 50% ethanol. Within 24 h, an acute inflammation occurred with hyperemia, edema, necrosis and an intense infiltration of granulocytes in the mucosa. The lesion proceeded into a T-lymphocyte/monocyte-driven chronic inflammation for two weeks and healed in 6 weeks. An acute inflammation recurred at the same site when the recovered animals were systemically injected with TNBS. We isolated RNA from colon tissue at 24 h, 1, 2, 4, 6 weeks after TNBS treatment and from the relapsed animals. The mRNA for cytokines IL-1beta, IL-6, IL-10 and the chemokines CINC, MIP-1alpha, MCP-1 were significantly (P < 0.05) elevated and persisted for 2 weeks, decreased in 6 weeks and increased again during relapse. IFN-gamma mRNA stayed at control levels initially, but increased dramatically in the second weeks of chronic inflammation as well as in relapse. The mRNA levels of adhesion molecules, ICAM-1, VCAM-1, the mucosal homing integrin beta7 as well as P- and E-selectin were greatly enhanced between 1 and 3 weeks. The data showed that the chronically inflamed tissue expresses a time-dependent changing pattern of TH1 cytokines and adhesion molecules that maintain the infiltration and activation of inflammatory cells and tissue injury.

Endogenous brain cytokine mRNA and inflammatory responses to lipopolysaccharide are elevated in the Tg2576 transgenic mouse model of Alzheimer's disease.

Beta-amyloid (Abeta) plaques have been shown to induce inflammatory changes in Alzheimer's disease brains. Cortical, but not cerebellar tissue from 16-month-old Tg2576 (Tg+) mice showed significant increases in interleukin (IL)-1alpha (2.2-fold), IL-1beta (3.4-fold), tumor necrosis factor-alpha (3.9-fold), and monocyte chemoattractant protein-1 (2.5-fold) mRNA levels compared to controls (Tg-). These changes were not apparent in 6-month-old Tg+ mice except for TNF-alpha. mRNA levels of glial fibrillary acidic protein and complement components, C1qA and C3 were also elevated in aged mice. Lipopolysaccharide (LPS) (25 microg/mouse, i.v.) induced a significantly greater production of IL-1beta protein in the cortices and hippocampi of Tg+ vs. Tg- mice at 1, 2, 4, and 6 h. Experiments in 6-month-old mice showed that not only was there less cytokine produced compared to 16-month-old mice, but the exacerbated cytokine response to LPS in Tg+ mice was not apparent. Higher levels of Abeta1-40 were measured in the cortices of 6- and 16-month-old Tg+ mice at 4-6 h after LPS, which returned to baseline after 18 h. We demonstrate that Abeta plaques elicit inflammatory responses in Tg2576 mice that are further exacerbated when challenged by an exogenous inflammatory insult, which may serve to amplify degenerative processes.

Effects of skyrin, a receptor-selective glucagon antagonist, in rat and human hepatocytes.

Peptidic glucagon antagonists have been shown to lower blood glucose levels in diabetic models (1-3), but attempts to identify small molecular weight glucagon receptor-binding antagonists have met with little success. Skyrin, a fungal bisanthroquinone, exhibits functional glucagon antagonism by uncoupling the glucagon receptor from adenylate cyclase activation in rat liver membranes (1). We have examined the effects of skyrin on cells transfected with the human glucagon receptor and on isolated rat and human hepatocytes. The skyrin used was isolated from Talaromyces wortmanni American Type Culture Collection 10517. In rat hepatocytes, skyrin (30 micromol/l) inhibited glucagon-stimulated cAMP production (53%) and glucose output (IC50 56 micromol/l). There was no detectable effect on epinephrine or glucagon-like peptide 1 (GLP-1) stimulation of these parameters, which demonstrates skyrin's selective activity. Skyrin was also evaluated in primary cultures of human hepatocytes. Unlike cell lines, which are largely unresponsive to glucagon, primary human hepatocytes exhibited glucagon-dependent cAMP production for 14 days in culture (EC50 10 nmol/l). Skyrin (10 micromol/l) markedly reduced glucagon-stimulated cAMP production (55%) and glycogenolysis (27%) in human hepatocytes. The inhibition of glucagon stimulation was a specific property displayed by skyrin and oxyskyrin but not shared by other bisanthroquinones. Skyrin is the first small molecular weight nonpeptidic agent demonstrated to interfere with the coupling of glucagon to adenylate cyclase independent of binding to the glucagon receptor. The data presented in this study indicate that functional uncoupling of the human glucagon receptor from cAMP production results in metabolic effects that could reduce hepatocyte glucose production and hence alleviate diabetic hyperglycemia.

Purified Chick Embryo Cell Culture Rabies Vaccine: interchangeability with Human Diploid Cell Culture Rabies Vaccine and comparison of one versus two-dose post-exposure booster regimen for previously immunized persons.

The equivalence and interchangeability of Purified Chick Embryo Cell Culture Rabies Vaccine (PCECV) to Human Diploid Cell Culture Rabies Vaccine (HDCV) and the immunogenicity of a reduced post-exposure regimen with PCECV was investigated. Statistical analyses revealed no difference (P

Flow cytometric detection of antigen-specific cytokine responses in lung T cells in a murine model of pulmonary inflammation.

Multiparameter flow cytometry was used to examine the cytokine responses of antigen-specific T lymphocytes isolated from the lungs of antigen-sensitized mice which developed pulmonary inflammation after aerosol challenge with ovalbumin (OA) (OA/OA). Lung T cells were stimulated in vitro with OA and anti-CD28 monoclonal antibody (mAb) in the presence of the secretion inhibitor, brefeldin A. T cell subsets were examined for intracellular cytokine expression using fluorochrome-labeled cell-surface specific and anti-cytokine antibodies. Antigen-specific responses resulted in significant numbers of CD4+ lung cells expressing cytoplasmic interleukin (IL)-2 (6%), IL-4 (1.5%), IL-5 (4%), and tumor necrosis factor (TNF)-alpha (11%), but not interferon (IFN)-gamma. Dual cytokine analyses demonstrated antigen-specific responses resulted in CD4+ T cells being positive for IL-2 and IL-4 or IL-2 and IL-5. TNF-alpha was the only antigen-specific cytokine response detected in CD8+ lung T cells after in vitro activation with OA. Cytokines in the supernatants of cultures activated with OA and anti-CD28 were measured by ELISA and the results confirmed the antigen-specific responses measured by flow cytometry. Polyclonal activation of lung T cells from OA/OA mice with 12-myristate 13-acetate (PMA), ionomycin, anti-CD3 mAb, and anti-CD28 mAb resulted in higher percentages of IL-2+ (43%) and IL-5+ (7%) CD4 cells when compared to CD4+ T cells from non-OA sensitized, challenged mice. CD8+ cells from OA/OA mice demonstrated intracellular staining for IL-2 (26%), TNF-alpha (55%), and IFN-gamma (37%), but not IL-4 or IL-5, after polyclonal activation. There is less agreement between intracellular cytokine staining of CD4+ T cells and cytokines released into the culture medium after polyclonal activation. Dual cytokine analyses of polyclonal-activated CD4+ cells demonstrated co-expression of IFN-gamma with IL-2, IL-4, or IL-5. T cells co-expressing IL-2 with IL-4 or IL-5 were also detected. These results demonstrate the utility of multiparameter flow cytometry to directly measure antigen-specific cytokine responses in subsets of T lymphocytes isolated from inflammatory sites.

Preclinical evaluation of anti-inflammatory activities of the novel pyrrolopyrimidine PNU-142731A, a potential treatment for asthma.

The anti-inflammatory properties of a novel pyrrolopyrimidine, PNU-142731A, in a murine model of antigen-induced eosinophilic lung inflammation are described. PNU-142731A, when given orally, demonstrated a dose-related inhibition of eosinophil- and lymphocyte-rich accumulation in the airways of ovalbumin (OA)-sensitized and challenged (OA/OA) C57BL/6 mice. The magnitude of the suppression of lung inflammation was also dependent on length of treatment. Reductions in the levels of interleukin (IL)-5, IL-6, and IgA in the bronchoalveolar lavage fluid of treated OA/OA mice, when compared with vehicle-sensitized control mice (V/OA), were observed. Plasma concentrations of IL-5, total IgE, and OA-specific IgG1 were also lowered in OA/OA mice by treatment. Histological assessment of formalin-fixed lung tissue sections confirmed that the compound blocked the accumulation of eosinophils in the airway tissue. Furthermore, significantly less mucus glycoproteins were seen in the lungs of PNU-142731A-treated OA/OA mice. Reverse transcription-polymerase chain reaction of lung tissue from PNU-142731A-dosed OA/OA mice demonstrated that mRNA for Th2 cytokines was less than that in vehicle-treated OA/OA controls. OA-elicited production of IL-4 by disaggregated lung tissue cells from PNU-142371A-treated OA/OA mice was also less than that of controls. In contrast, the release of Th1 cytokines (IL-2 and interferon-gamma) were elevated. Similarly, the OA-stimulated release of Th2 cytokines (IL-5 and IL-10) by splenocytes from PNU-142731A-treated OA/OA mice were inhibited. Combined therapy of OA/OA mice with PNU-142731A and suboptimal doses of dexamethasone revealed that PNU-142731A had steroid-sparing effects. These characteristics of PNU-142731A in a murine model of allergic tissue inflammation support its clinical development as a potential treatment for asthma.

Involvement of intercellular adhesion molecule-1 in the antigen-induced infiltration of eosinophils and lymphocytes into the airways in a murine model of pulmonary inflammation.

We investigated the effects of in vivo intraperitoneal treatment with the rat monoclonal antibody (mAb), YN1.7.4 (YN1) against intercellular adhesion molecule-1 (ICAM-1) on the ovalbumin (OA)-inhalation-induced infiltration of leukocytes into the airways of OA-sensitized mice. YN1 (100 to 400 microg) given over a period of 72 h dose-dependently reduced the influx of lymphocytes and eosinophils into the bronchial lumen by > 60% and > or = 70%, respectively, when compared with saline or purified rat IgG-treated controls. Alveolar macrophages (AM) in the bronchoalveolar lavage fluid (BALF) were also decreased by > 50%. Lung tissue inflammation as determined by histopathologic examination was reduced. The number of neutrophils in the blood of OA-sensitized mice 3 days after challenge was significantly increased by treatment with YN1. However, at 24 h and 72 h after OA-challenge, the numbers of eosinophils and mononuclear cells in the bone marrow were reduced by YN1 treatment. Additionally, at 72 h after OA-challenge, the numbers of bone-marrow neutrophils were depressed. BALF levels of interleukin-5 (IL-5) and of IgA were lower for YN1-treated mice than for controls. With increasing doses of YN1, the levels of anti-ICAM-1 mAb in the plasma were proportionally increased. To correlate these results with YN1 treatment, blood and BALF T cells and BALF eosinophils were examined with flow cytometry. Blood T cells from YN1-treated mice were unable to bind phycoerythrin (PE)-labeled anti-ICAM- mAb ex vivo. These results implied that ICAM-1 on these cells was bound (occupied) by YN1 administered in vivo. Dose-related decreases were observed in the percentage and mean channel fluorescence (MCF) values of ICAM-1+ BALF T cells and eosinophils. The percentages of CD11a+ or CD49d+ eosinophils were also suppressed. Our data suggest that ICAM-1 is an important molecule involved in the recruitment of leukocytes into the airways of sensitized mice after pulmonary challenge.

Roles of adhesion molecules ICAM-1 and alpha4 integrin in antigen-induced changes in microvascular permeability associated with lung inflammation in sensitized brown Norway rats.

Increased microvascular permeability and mucosal edema are pathological features of airway inflammation in asthma. In this study, we investigated the characteristics of the edema response occurring in a model of antigen-induced lung inflammation in sensitized brown Norway rats and examined the effects of monoclonal antibodies (mAbs) to adhesion molecules on this response. Ovalbumin (OA) challenge-induced increases in lung permeability were determined by the leakage of 125I-labeled bovine serum albumin (BSA) into the extravascular tissues of the lungs 24 h after challenge in animals intravenously injected (prechallenge) with this tracer. Inflammatory cell infiltration into the alveolar space was determined by bronchoalveolar lavage (BAL). Mean extravascular plasma volume in the lung increased 233% as compared with control (P < 0.005) at 24 h and increased to 517% by 72 h. The 24-h edema response was completely inhibited by two oral doses (0.1 mg/kg) of dexamethasone 1 h before, and 7 h after, challenge. Intraperitoneal administration of the anti-rat ICAM-1 mAb 1A29, or anti-rat alpha4 integrin mAb TA-2 (2 mg/kg at 12 and 1 h before, and 7 h after, antigen challenge), significantly suppressed eosinophil infiltration into the alveolar space without inhibiting the enhanced microvascular leakage and lung edema. Determination of plasma antibody concentrations by ELISA of mouse IgG1 indicated that sufficient concentrations of the appropriate mAb were present to block alpha4- or ICAM-1-dependent adhesion. The results suggest that increases in microvascular permeability and plasma leakage occurred independently of eosinophil accumulation.

Intercellular adhesion molecule-1-deficient mice have antibody responses but impaired leukocyte recruitment.

The role of intercellular adhesion molecule-1 (ICAM-1) in murine lung inflammation was examined in vivo. Ovalbumin (Ova)-sensitized and -challenged ICAM-1-deficient (KO) mice had decreased accumulation of leukocytes in the bronchoalveolar lavage fluid compared with wild-type (WT) mice. Lung tissue inflammation was also attenuated. Ova immunization and challenge produced equivalent plasma levels of Ova-specific immunoglobulin (Ig) G1 and higher concentrations of IgE in KO versus WT mice. Ova-dependent induction of cytokines in vitro, as measured by enzyme-linked immunosorbent assay, was impaired in splenocytes from KO mice compared with the comparable release of interleukin (IL)-5 and IL-10 from anti-CD3-stimulated WT and KO splenocytes. Methacholine-induced increases in trapped gas in lungs of Ova-sensitized and -challenged WT mice were greater than those of KO mice. The activation of lung tissue nuclear factor-kappa B was diminished in KO mice after Ova provocation. This suggests that ICAM-1 was important for activation of the inflammatory cascade leading to the recruitment of leukocytes but was not critical for the generation of antibody responses in vivo.

Identification of cytokine and adhesion molecule mRNA in murine lung tissue and isolated T cells and eosinophils by semi-quantitative reverse transcriptase-polymerase chain reaction.

We have used a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay to detect the expression of mRNA for inflammatory cytokines, integrins and selectins in murine lung tissue, and T cells and eosinophils isolated from lung and bronchoalveolar lavage (BAL) fluid in an in vivo model of ovalbumin (OA)-induced airway inflammation. RNA was isolated from whole lung tissue at 1, 6, 24, 48, 72 h, and 7 days after OA inhalation. mRNA for the Th2 cytokines, IL-4, -5, -6, -10 and -13 in OA-sensitized mice were significantly elevated compared with non-sensitized mice. IL-2, TNF-beta, and eotaxin mRNA were also increased, but IFN-gamma mRNA was not. P- and E-selectin mRNA levels were also enhanced in lung tissue between 6 and 72 h after challenge. Lung T cells were isolated by cell sorting with a flow cytometer at 3, 12, 24, 48 and 72 h after challenge. mRNA levels for IL-5 and -10 were greater in T cells from OA-sensitized and -challenged mice than controls at 24 h. BAL fluid from OA-sensitized and -challenged mice also had significantly higher IL-5 levels than controls. BAL fluid T cells and eosinophils were obtained at 48 and 72 h after aerosol challenge and were purified by cell sorting. Messenger RNA for IL-1 alpha, -2, -4, -5, -10, IFN-gamma, and beta 1 were detected in T cells at both time points. Transcripts for IL-1 alpha, -4, -5, -13, TNF-alpha and beta, and alpha 4, beta 1 and beta 7 were also identified in BAL eosinophils. These data show that in addition to murine lung T cells, airway eosinophils may also contribute to the inflammatory response by their ability to express mRNA for cytokines and integrins.

Airway recruitment of leukocytes in mice is dependent on alpha4-integrins and vascular cell adhesion molecule-1.

The involvement of the alpha4-integrin very late activation antigen 4 and vascular cell adhesion molecule-1 (VCAM-1) in leukocyte trafficking into the airways of ovalbumin (OA)-sensitized and OA-challenged mice was investigated using in vivo administration of anti-alpha4 monoclonal antibodies (mAb) PS/2, R1-2, and M/K-2.7 (MK2), specific for VCAM-1. VCAM-1 was upregulated on endothelial cells in lung tissue after OA inhalation. PS/2, R1-2, or MK2 significantly inhibited the recruitment of eosinophils and lymphocytes into the bronchoalveolar lavage (BAL) fluid and decreased inflammation in the lung tissues. Escalating in vivo doses of PS/2 or MK2 increased circulating levels of rat immunoglobulin G in the plasma. The binding of phycoerytherin-labeled anti-alpha4 mAb to blood T cells from PS/2-treated mice was reduced, implying that alpha4 sites were already occupied. T cells and eosinophils in BAL fluid from mice treated with PS/2 or MK2 were phenotypically different from controls. Selective decreases of alpha4+ T cells in the BAL fluid after PS/2 or MK2 treatment were coupled with changes in CD8+, CD11a, and CD62L expression. The alpha4-integrin and VCAM-1 may have important roles in the antigen-induced recruitment of T cells and eosinophils during OA-induced airway inflammation. The data suggest that these adhesion molecules may be suitable targets for therapeutic intervention in certain conditions of pulmonary inflammation.

Pulmonary ferritin: differential effects of hyperoxic lung injury on subunit mRNA levels.

Ferritin is an iron storage protein that is regulated at the transcriptional and transcriptional levels, resulting in a complex mixture of tissue- and condition-specific isoforms. The protein shell of ferritin is composed of 24 subunits of two types (heavy or light), which are encoded by two distinct and independently regulated genes. In the present studies, the isoform profile for lung ferritin differed from other tissues (liver, spleen, and heart) as determined by isoelectric focusing (IEF) and polyacrylamide gel electrophoresis (PAGE). Lung ferritin was composed of equal amounts of heavy and light subunits. Differences in isoform profiles were the result of tissue-specific differential expression of the ferritin subunit genes as demonstrated by Northern blot analyses. Like heart ferritin, lung ferritin exhibited a low iron content that did not increase extensively in response to iron challenge, which contrasts with ferritins isolated from liver or spleen. When animals were exposed to hyperoxic conditions (95% oxygen for up to 60 h), ferritin heavy subunit mRNA levels did not markedly change at any of the investigated time points. In contrast, ferritin light subunit mRNA increased severalfold in response to hyperoxic exposure. Investigation of the cytoplasmic distribution of ferritin mRNA showed that a substantial portion was associated with the ribonucleoprotein (RNP) fraction of the cytosol, suggesting that a pool of untranslated ferritin mRNA exists in the lung. Upon hyperoxic insult, all ferritin light subunit mRNA pools (RNP, monosomal, polysomal) were elevated, although a specific shift from RNP to polysomal pools was not evident. Therefore, the increase in translatable ferritin mRNA in response to hyperoxia resulted from transcriptional rather than specific translational activation. The observed pattern of light chain-specific transcriptional induction of ferritin is consistent with the hypothesis that hyperoxic lung injury is at least partially iron mediated.

Cytokine networks in allergic lung inflammation: an opportunity for drug intervention.

Eosinophils and mast cells have long been considered as the major effector cells ultimately responsible for bronchial obstruction and airway hyper-responsiveness in asthmatics. However, there is now accumulating evidence that products of Th2 lymphocytes may orchestrate the generation, accumulation, and activation of these cells within the airway wall. Since the first report by Mosmannet al. in 1986 that murine helper T-cell clones could be divided into two subsets, Th1 and Th2, depending on their pattern of cytokine secretion, and observations that polarisation of Th1- or Th2-dependent cytokine production could be correlated with distinct autoimmune and allergic disorders, there has been an increasing interest in the possibility that pharmacological manipulation of the Th1/Th2 paradigm could provide novel treatments for human disease. This review summarises the evidence to date, attempts to explain some apparent discrepancies, and indicates opportunities for therapeutic intervention.

Safety and immunogenicity of Lyssavac Berna human diploid cell rabies vaccine in healthy adults.

A clinical trial testing the safety and immunogenicity of a newly developed human diploid cell rabies vaccine (Lyssavac-HDC) was conducted on subjects at three colleges of veterinary medicine in the United States. Lyssavac-HDC is a sterile lyophilized vaccine containing no antibiotics or preservatives and is administered intramuscularly as a 0.5 ml dose of vaccine containing at least 2.5 i.u. of rabies inactivated antigen per dose. Subjects were given either a three dose pre-exposure series (days 0, 7, and 28), followed by one booster dose of vaccine (day 360); or a five dose simulated post-exposure series of injections (days 0, 3, 7, 14, and 28). All subjects in the post-exposure and pre-exposure groups possessed adequate levels of rabies neutralizing antibody (> or = 5) when tested on day 14 and day 28, respectively. Subjects in the pre-exposure group demonstrated a vigorous anamnestic response after the administration of one booster dose of vaccine on day 360. The type and severity of local and systemic reactions observed were comparable to other primary cell culture rabies vaccines. Significantly, there were no type III hypersensitivity reactions reported in subjects previously immunized with Lyssavac-HDC after the administration of a booster dose of vaccine on day 360.

Role of very late activation antigen-4 in the antigen-induced accumulation of eosinophils and lymphocytes in the lungs and airway lumen of sensitized brown Norway rats.

We used flow cytometry and treatment in vivo with a monoclonal antibody (mAb), TA-2, to the alpha 4 integrin to investigate the role of alpha 4 beta 1, CD49d/CD29 (VLA-4) in antigen-induced lung inflammation in Brown Norway (BN) rats. Ovalbumin (OVA) inhalation induced an accumulation of eosinophils and lymphocytes in the lungs and bronchoalveolar lavage (BAL) fluid of sensitized BN rats at 24 h after challenge. Phenotypic analyses demonstrated that the percentages of T cells expressing detectable alpha 4 and CD25 in the bronchial lumen after antigen challenge were dramatically increased compared with blood and lymph node T cells. The mean channel fluorescence values of alpha 4 expression were also increased on BAL T cells compared with blood or lymph node T cells. Treatment of OVA-sensitized rats in vivo with total cumulative doses of 0.75 to 6 mg/kg TA-2 mAb intraperitoneally produced dose-related increases in circulating TA-2 and a peripheral blood lymphocytosis, basophilia, and eosinophilia. Flow cytometric analysis of the peripheral blood T cells after in vivo TA-2 mAb administration showed decreases in detectable alpha 4 when these cells were examined ex vivo. Treatment with TA-2, but not an isotype-matched control mouse immunoglobulin G1 mAb, markedly inhibited the OVA-induced recruitment of lymphocytes and eosinophils into the airway lumen. Very few CD3+CD49d+ cells migrated into BAL fluid following anti-alpha 4 mAb treatment in vivo. Treatment with TA-2 also significantly attenuated OVA-induced inflammatory histopathology. We conclude that VLA-4 is a critically important adhesion molecule involved in antigen-specific lung inflammation in sensitized BN rats.

Role of intercellular adhesion molecule-1 in antigen-induced lung inflammation in brown Norway rats.

We investigated the involvement of intercellular adhesion molecule-1 (ICAM-1; CD54) in ovalbumin (OA) antigen-induced lung inflammation in sensitized Brown Norway (BN) rats by using flow cytometry and in vivo treatment with a murine monoclonal antibody (MAb), 1A29, directed against rat ICAM-1. OA-challenge induced an eosinophil and lymphocyte-rich accumulation of leukocytes into the airway lumen. Between 75 and 90% of the T cells in bronchoalveolar lavage (BAL) fluid after challenge expressed CD54 and CD11a and were of the memory phenotype. 1A29 treatment produced dose-related increases in circulating 1A29 and blood neutrophils. In the BAL fluid of 1A29-treated animals, significant (P < 0.05) reductions in the numbers of eosinophils and lymphocytes, but not neutrophils or alveolar macrophages, were observed in association with a reduced inflammatory pathology in lung tissue. 1A29 administration reduced the number of detectable ICAM-1 binding sites on T cells in peripheral blood and BAL fluid examined ex vivo by flow cytometry. We conclude that ICAM-1 is critically important for the antigen-specific recruitment of eosinophils and lymphocytes into the lungs.

Phenotypic analysis of airway eosinophils and lymphocytes in a Th-2-driven murine model of pulmonary inflammation.

In order to investigate whether the pulmonary response to helminth antigens mimics that seen in allergic inflammation of the airways, we have examined the phenotypic characteristics of lymphocytes and eosinophils recruited to the airways following Nippostrongylus brasiliensis (N.b.) infection. Specifically, the cellular response was divided into an early and a late phase. During the early response there was a small but significant increase in neutrophil numbers recovered by bronchoalveolar lavage (BAL). Phenotypic analysis of BAL leukocytes revealed an early rise in the percentage of BAL lymphocytes expressing the naive T cell markers CD45RB and L-selectin, and the activation marker IL-2R. In addition, during the early response, there was an increased percentage of lymphocytes expressing the gamma delta TCR, but not the alpha beta TCR. In contrast, the late response was marked by a much larger accumulation, in the lungs and BAL, of memory CD4+ T lymphocytes and an influx of small, hypodense eosinophils which produced LTB4 and LTC4 on stimulation with calcium ionophore. At this time there was a substantial increase in the number of T lymphocytes and eosinophils expressing ICAM-1 and the integrins VLA-4 and LFA-1, implicating these adhesion molecules in inflammatory cell recruitment to the airways. We conclude that the pattern and phenotypic characteristics of the cellular recruitment seen following N.b. infection resemble those seen in early- and late-phase allergic inflammation of the airways in asthma, and therefore N.b. may be used to model these aspects of the disease.

Phenotypic characterization of T lymphocytes emigrating into lung tissue and the airway lumen after antigen inhalation in sensitized mice.

Cytokines released from CD4+ T lymphocytes contribute to the pathogenesis of asthma by influencing the differentiation and function of eosinophils, the primary effector cells that cause airway epithelial damage. Using a model of ovalbumin (OA)-induced, eosinophil-rich chronic lung inflammation in sensitized mice, we have defined the role of T lymphocytes further by using three-color flow cytometry to characterize the adhesion and activation antigens that may be associated with the migration of these cells into the lung and airway lumen. OA inhalation in OA-sensitized C57BL/6 mice resulted in an early (6 to 24 h) influx of neutrophils into the bronchial lumen as enumerated by bronchoalveolar lavage (BAL), which was followed by a marked accumulation of lymphocytes and eosinophils between 24 to 72 h. Phenotypic analysis of BAL or lung tissue T cells showed that most Thy-1 CD3+ T cells were CD4+ (CD4: CD8 ratio of 3 to 4:1). The majority (90%) of the T cells in lung or BAL fluid expressed alpha beta T-cell receptors (TCR). Only 3 to 7% of the T cells were gamma delta TCR+ even though almost 25% of the T cells were CD4- CD8-. There were very few natural killer (NK) or B cells in BAL fluid compared with 15% B cells in dissagregated lung tissue. In contrast to T cells in spleen, almost all the lung and BAL T cells were of the memory phenotype, as ascertained by the expression of high levels of CD44 and by the absence of L-selectin and CD45RB on the cell surface. Fifty to ninety percent of lung and BAL T cells from vehicle-sensitized or OA-sensitized and challenged mice expressed the adhesion molecules CD11a (LFA-1), CD54 (ICAM-1), and CD49d (VLA-4). The early T-cell activation marker CD69 was upregulated on 30% of the lung and BAL T cells in OA-sensitized mice after antigen inhalation. When BAL fluid T cells from OA-sensitized and challenged mice were analyzed for their coexpression of adhesion and/or activation molecules, 75% of the cells that expressed one of three adhesion molecules, CD54, CD49d, or CD11a, also expressed at least one of the other two antigens. At least 15% of BAL T cells had all three of these molecules on their cell surfaces. The OA-dependent, temporally regulated emigration of T cells into the bronchial lumen after exposure to aerosolized antigen may be correlated with the accumulation of cells that express the memory phenotype with enhanced expression of adhesion molecules.