Localization of nitric oxide synthases and nitric oxide production in the rat mammary gland

We investigated nitric oxide (NO) production and the presence of nitric oxide synthase (NOS) in the mammary gland by use of an organ culture system of rat mammary glands. Mammary glands were excised from the inguinal parts of female Wistar-MS rats primed by implantation with pellets of 17beta-estradiol and progesterone and were diced into approximately 3-mm cubes. Three of these cubes were cultured with 2 ml of 10% FCS/DMEM plus carboxy-PTIO (an NO scavenger, 100 microM) in the presence or absence of LPS (0.5 microgram/ml) for 2 days. The amount of NO produced spontaneously by the cultured mammary glands was relatively minute at the end of the 2-day culture period, and the NO production was significantly enhanced by the presence of LPS. This enhancement of NO production was completely eliminated by addition of hydrocortisone (3 microM), an inhibitor of inducible NOS (iNOS), to the incubation medium. Immunoblot analyses with specific antisera against NOS isoforms such as iNOS, endothelial NOS (eNOS), and brain NOS (bNOS) showed immunoreactive bands of iNOS (122 +/- 2 kD) and eNOS (152 +/- 3 kD) in extracts prepared from the mammary glands in the culture without LPS. The immunoreactive band of iNOS was highly intense after the treatment of mammary glands with LPS, whereas the corresponding eNOS immunoreactive band was faded. The immunohistochemical study of anti-iNOS antiserum on frozen sections of the cultured mammary glands showed that an immunoreactive substance with the antiserum was localized to the basal layer (composed of myoepithelial cells of alveoli and lactiferous ducts) of the mammary epithelia and to the endothelium of blood vessels that penetrated into the interstitium of the mammary glands. Histochemical staining for NADPH-diaphorase activity, which is identical to NOS, showed localization similar to that of iNOS in the mammary glands. Similar observations were noted in the immunohistochemistry of eNOS. In contrast, the immunoreactive signal with the bNOS antiserum was barely detected in the epithelial parts of alveoli and lactiferous ducts of the mammary glands. These observations demonstrate that three isoforms of NOS are present not only in the endothelium of blood vessels but also in the parenchymal cells (the glandular epithelium) of the rat mammary gland, such as epithelial cells and myoepithelial cells, and suggest that NO may have functional roles in the physiology of the mammary glands.     

Lymphocyte subpopulations in the mammary gland of the goat

Mammary glands of pregnant, lactating and resting goats were studied by immunohistochemistry for lymphocyte subpopulations using a panel of monoclonal antibodies. All T lymphocyte subpopulations that may have a role in the immune response, CD2+, CD4+, CD8+ and gamma delta T cells and subsets, were present in the mammary gland and were noted to increase in number progressively during pregnancy, decrease significantly during lactation, and then moderately increase during the resting period. CD4+ cells, the predominant cell type in the mammary gland, were located mainly in the connective tissue, whereas CD2+, CD8+ and TcR1-N24+ cells were predominant in the intraepithelial areas. TcR1-N6+ cells were detected almost exclusively during pregnancy, being localized mainly in the connective tissue. Their proportion decreased markedly following parturition. Very few WC1-N3+ and -N4+ cells were detected in the mammary gland. It is suggested that the majority of gamma delta T lymphocytes in the mammary gland of the goat are CD2+ CD8+ WCl-, a distinctive subset from that of the WCl+ subset in peripheral blood.     

Inflammatory cells in the bronchial glands of smokers with chronic bronchitis

To characterize the inflammatory process in the bronchial glands of smokers with chronic sputum production, we examined lobar bronchi from 18 subjects undergoing lung resection for localized pulmonary lesions, all with a history of cigarette smoking. Nine of the subjects had symptoms of chronic bronchitis and chronic airflow obstruction, and nine were asymptomatic, with normal lung function. The number of neutrophils, eosinophils, mast cells, macrophages, CD4+ and CD8+ T-lymphocytes, and the ratio of CD4+ to CD8+ cells were assessed in the bronchial glands, epithelium, and submucosa. Cells were identified through immunohistochemistry. Smokers with symptoms of chronic bronchitis had an increased number of neutrophils (p = 0.01) and macrophages (p = 0.03) and a decreased CD4+/CD8+ ratio (p = 0.01) in the bronchial glands as compared with asymptomatic smokers. Chronic bronchitic smokers also had an increased number of epithelial neutrophils (p = 0.04), whereas the numbers of macrophages and CD4+ and CD8+ T-lymphocytes in the epithelium and submucosa were similar in the two groups of smokers. No differences in numbers of eosinophils or mast cells were observed between bronchitic and asymptomatic smokers in any of the compartments examined. In conclusion, smokers with chronic sputum production have an increased infiltration of neutrophils and macrophages and an increased proportion of CD8+ T-lymphocytes in their bronchial glands, supporting the important role of bronchial-gland inflammation in the pathogenesis of chronic bronchitis.     

Congenital diaphragmatic hernia--a review of pre- and postoperative chest radiology

Delayed-type hypersensitivity responses against bovine leukemia virus (BLV) envelope glycoprotein (gp60) were induced in the skin of sheep vaccinated with recombinant vaccinia virus (RVV) expressing BLV glycoprotein. The lesions were characterized by marked infiltration of lymphocytes, slight migration of neutrophils, eosinophils, and macrophages in the dermis to hypodermis, and partial intercellular edema in the reticular layer. Immunohistochemical analysis with monoclonal antibodies demonstrated that the lymphocytic infiltrates consisted mainly of CD8+ T cells (53.7-55.8% at 48 hours post-challenge of BLV), CD4+ T cells (24.7-26.7%), and B cells (11.5-16.9%). The role of CD4+ and CD8+ T cells in suppressing BLV growth in RVV-vaccinated animals is discussed.     

Macrophage-parasite relationship in theileriosis. Reversible phenotypic and functional dedifferentiation of macrophages infected with Theileria annulata

Theileria annulata is a tick-transmitted protozoan parasite of cattle, which transforms cells of macrophage (Mphi) or B cell lineage. Bone marrow cells, bone marrow cell-derived, and monocyte-derived Mphi were infected with T. annulata sporozoites, and the resulting cell lines were assessed for surface marker expression and function. Transformed lines expressed histocompatibility complex (MHC) class-I and II, CD44, CD45, and the myeloid marker DH598-surface markers CD14, CD11b, M-M7, TH57A, and to a lesser extent CD11a/CD18, CD11c, and ACT(B), were down-regulated. Likewise, transformed cells failed to express Mphi functions (Fc-receptor-mediated phagocytosis, phorbol myristate acetate-induced oxidative burst, lipopolysaccharide-induced tumor necrosis factor alpha, and nitric oxide generation and procoagulant activity up-regulation). Mphi origin was assured by homogeneity of the starting population, cloning of cells by limiting dilution, and repeated microscopic and flow cytometric monitoring of the cell lines. Elimination of the parasite by treatment with BW720c resulted in the re-acquisition of monocyte lineage properties, as evidenced by up-regulation of CD14, and by re-acquisition of the capacity to ingest opsonized sheep red blood cells and bacteria. Thus, Mphi transformed by T. annulata appear to undergo a process of parasite-induced dedifferentiation but reassume the differentiated phenotype upon elimination of the parasite.     

Chemokine-dependent upregulation of CD11b on specific leukocyte subpopulations in human whole blood: effect of anticoagulant on rantes and MIP-1 beta stimulation

The effect of anticoagulant (heparin vs EDTA) on chemokine induced CD11b upregulation on neutrophils, eosinophils, and monocytes in human whole blood was determined. For most of the chemokines (IL-8, GRO-alpha, MCP-1, MIP-1 alpha) the difference in the response of leukocytes in EDTA anticoagulated blood vs those in heparinized blood was the degree of their maximal response, with a slightly higher maximal increase in CD11b expression usually seen in cells from EDTA anticoagulated blood. Two chemokines were exceptions to this: RANTES and MIP-1 beta. RANTES is considered to be a stimulator of monocytes and eosinophils and not of neutrophils. As expected, neutrophils in heparinized whole blood did not respond to RANTES; however, neutrophils in EDTA anticoagulated blood had a significant increase in CD11b when exposed to high concentrations (1 microM) of RANTES. RANTES-induced CD11b expression on monocytes and eosinophils in these samples were the same in either heparin or EDTA. In EDTA anticoagulated blood, MIP-1 beta did not elicit a response in either monocytes, eosinophils or neutrophils; however, in heparinized blood, all three cell types increased CD11b expression upon exposure to 1 microM MIP-1 beta.     

The response of gamma delta T cells to Plasmodium falciparum is dependent on activated CD4+ T cells and the recognition of MHC class I molecules

Peripheral blood gamma delta T cells from non-exposed individuals respond to antigens of the malaria parasite, Plasmodium falciparum, in vitro. This response, largely caused by T cells bearing the V gamma 9+ chain of the T-cell receptor, is stimulated by components of the parasite expressed on the schizont stage and released at schizont rupture. The response of V gamma 9+ T cells to parasite components is inhibited by antibodies to major histocompatibility complex (MHC) class I and class II. However, the inhibition by anti-MHC class II antibodies can be overcome by the addition of interleukin-2 (IL-2) to the cultures, suggesting that gamma delta T cells themselves do not recognize MHC class II molecules but require an MHC class II-dependent response taking place in the culture. In contrast, the inhibition by anti-class I antibodies cannot be reversed by addition of IL-2. Since an accompanying CD4+ T-cell response occurred in peripheral blood mononuclear cells cultured with P falciparum antigens, it was considered that these cells provide the cytokines necessary for the subsequent activation and expansion of V gamma 9+ T cells recognizing components of the parasite and MHC class I molecules. This was confirmed by reconstituting the response of enriched gamma delta T cells to P falciparum schizont extract by addition of purified CD4+ T cells.     

Beta-turn induction by C60-based fulleroproline: synthesis and conformational characterization of Fpr/Pro small peptides

In this paper, we examined the induction of autoimmune-like histologic changes in the liver and other organs of mice undergoing graft-versus-host reaction (GVHR) with MHC class I disparity by the administration of bacterial lipopolysaccharide (LPS), on the assumption that stimulation with LPS could be an exacerbating factor. Spleen cells of C57BL/6 (B6) mice were injected twice into (B6 x bml) F1 recipient mice at an interval of 7 days to induce MHC class I GVHR and then challenged with 1 microg of LPS intravenously on the next day of the cell transfer. The hepatic lesions of the group of MHC class I GVHR mice challenged with LPS showed marked cellular infiltration at the portal area and focal necrosis was observed in the hepatic lobule. The major infiltrating cells were CD8+, and others including CD4+ cells being of minor populations. In addition, ductal lesions in extrahepatic organs, including the pancreas and salivary glands also showed marked cellular infiltration. Thus, we have demonstrated that LPS induced ductal lesions in mice with MHC class I disparity. CD8+ cells were detected at the destructive hepatic lesions, which might be effector cells. These findings indicate that LPS might be one of the potential factors which augment autoimmune-like lesions.     

Sonographic analysis of recurrent parotitis in children: a comparative study with sialographic findings

BACKGROUND: Patients with asthma show altered surface expression of the adhesion molecules CD11b and L-selectin on airway granulocytes compared with blood granulocytes. OBJECTIVE: To investigate whether this modulation is related to disease activity or due to transendothelial migration, we compared the CD11b and L-selectin expression on blood and induced sputum eosinophils and neutrophils between patients with asthma and normal subjects. METHODS: Eleven normal subjects (21-43 years), nine patients (21-34 years) with mild atopic asthma and 10 patients (20-47 years) with moderate to severe atopic asthma on regular treatment with inhaled steroids underwent sputum induction by inhalation of nebulized hypertonic saline (4.5%). CD11b and L-selectin expression on granulocytes from blood and DTT-homogenized sputum were analysed by flow cytometry. Eosinophils could be discriminated from neutrophils by using depolarized light scatter. Disease activity was assessed by baseline FEV1 and airway responsiveness to histamine (PC20). RESULTS: Sputum eosinophils showed higher expression of CD11b (P<0.001) and lower expression of L-selectin (P<0.001) compared with peripheral blood eosinophils. CD11b and L-selectin expression on eosinophils from blood or sputum did not differ between the three groups. Similar results were obtained for neutrophils. The PC20 in the patients with moderate-to-severe asthma was related to CD11b expression on blood (R=-0.92, P=0.001) and sputum eosinophils (R=0.75, P=0.02). CONCLUSIONS: Flow cytometry of induced sputum granulocytes from asthmatic as well as normal subjects is feasible. We conclude that the modulated expression of CD11b and L-selectin on airway granulocytes is not specific for asthmatic airway inflammation, but is probably the result of tissue migration per sé. This implies that CD11b and L-selectin expression on granulocytes in induced sputum cannot be used as marker of disease activity.     

Tumor necrosis factor alpha induces migration of human eosinophils

In order to evaluate the role of tumor necrosis factor alpha (TNF alpha) in the recruitment of eosinophils and neutrophils into the tissues, we studied the effect of TNF alpha on the migration of those cells in vitro, employing a modified Boyden's chamber technique. TNF alpha induced a significant migration of human eosinophils in a dose-dependent manner, and the preincubation of eosinophils with TNF alpha enhanced platelet activating factor (PAF)-induced eosinophil migration. Checkerboard analysis revealed that the eosinophil migration induced by TNF alpha was mainly due to chemokinesis. On the other hand, TNF alpha induced neither neutrophil migration nor enhancement of PAF-induced neutrophil migration. These results indicate that TNF alpha possesses a chemokinetic effect on human eosinophils and that TNF alpha augments the migration of eosinophils by PAF.     

Effect of theophylline on CD11b and L-selectin expression and density of eosinophils and neutrophils in vitro

The nonspecific phosphodiesterase inhibitor theophylline, widely used in asthma therapy, may cause a decrease in inflammatory responses of airways. In asthma, eosinophils migrate to the airway wall and become activated. Activated eosinophils are characterized by low cell density, as well as increased expression of CD11b and reduced expression of L-selectin, two adhesion molecules involved in transendothelial migration. To study the anti-inflammatory effect of theophylline on granulocyte adhesion molecules in vitro, the platelet-activating factor (PAF)-induced density shift was determined by density centrifugation and the modulation of CD11b and L-selectin expression by flow cytometry on eosinophils and neutrophils in human whole blood. A relatively high concentration of theophylline (10(-3) M) inhibited the increase in the percentage of hypodense eosinophils and neutrophils in whole-blood samples after PAF stimulation in vitro. A more pharmacological concentration (10(-4) M) inhibited the CD11b upregulation and L-selectin shedding induced by PAF (10(-7) M) on both eosinophils and neutrophils. The effect of isoproterenol on the inhibitory effect of theophylline was mainly additive, but a small synergistic effect could not be excluded. In conclusion theophylline can attenuate eosinophil and neutrophil activation in vitro at the level of adhesion molecule expression and changes in cell density. This may have implications for transendothelial migration of these cells in asthma.     

Mechanisms of eosinophil recruitment

As with other types of leukocytes, mechanisms that function to enable the recruitment of eosinophils into specific sites of immune reactions involve a complex and cumulative interplay of many molecules and pathways. No single chemoattractant is specific for eosinophils, but rather various chemoattractants active on eosinophils can also elicit migration of other specific cell types. Humoral mediators causing eosinophil migration include C5a and platelet-activating factor, whereas cytokines active as eosinophil chemoattractants include interleukin (IL)-2, IL-3, IL-5, granulocyte/macrophage colony-stimulating factor, lymphocyte chemoattractant factor, and RANTES. Eosinophils utilize several pathways to adhere to vascular endothelial cells, including binding to intercellular adhesion molecule-1, E-selectin, and vascular cell adhesion molecule-1 (VCAM-1). The lack of binding of neutrophils to VCAM-1 and the enhanced expression of VCAM-1 elicited by IL-4 contribute to preferential eosinophil accumulation. Eosinophil recruitment is dependent not only on ligands expressed on eosinophils and molecules inducible on endothelial cells but also on processes active during transendothelial migration and extravascular migration in the extracellular spaces.     

Immune responsiveness of nematode-resistant or susceptible Romney line-bred sheep to continuous infection with Trichostrongylus axei

Two divergent lines of Romney sheep have been selected on the basis of differences in faecal egg counts (FEC) to natural poly-generic parasite challenge in New Zealand. However, it is not known if the expression of resistance or susceptibility extends to parasitic nematodes that were not a major part of the selection challenge. To examine this, the immune response to infection with Trichostrongylus axei was examined in these sheep lines. Changes in the proportions of CD5+, CD4+, CD8+, T19+ and CD45R+ lymphocytes and parasite specific antibody titres in peripheral blood were monitored each week in six resistant and six susceptible line lambs that were maintained indoors in pens during the course of 14 weekly infections with 10,000 T. axei larvae. No difference in FEC was observed. Similarly, no significant difference in T. axei specific antibody titre between sheep lines was seen although antibody titre increased steadily from Week 4. Significant increases in the proportions of CD5+, CD4+, CD8+ and T19+ cells occurred in both resistant and susceptible line lambs during Weeks 1-4 of infection. Following peak levels, proportions of CD5+, CD4+ and CD8+ cells fell with the rate of decline of CD5+ and CD4+ cells significantly greater in the resistant line lambs. Proportions of CD45R+ cells showed significant changes with time that were the inverse of those of CD5+, CD4+ and CD8+ cells. Susceptible line lambs showed higher proportions of CD5+ and lower proportions of CD45R+ cells than resistant lambs before infection with T. axei. Overall, during infection, these differences were maintained while CD4+ and T19+ levels were higher in the susceptible line lambs.     

The role of gammadelta T lymphocytes in lipopolysaccharide-induced eosinophil accumulation into the mouse pleural cavity

LPS induces an accumulation of eosinophils in the pleural cavity that requires resident macrophages and lymphocytes, but is independent of IL-5 production. In the present study we investigated the involvement of different T lymphocyte subsets on the modulation of LPS-induced eosinophil accumulation into the pleural cavity of mice. Within 4 h after LPS injection the number of neutrophils in the pleural cavity increased significantly. Mononuclear cell counts increased after 12 h, while a significant rise on eosinophil counts was observed only after 24 h. T lymphocytes counts were increased in the pleural cavity 24 and 48 h after LPS administration. This T lymphocyte accumulation was accounted for by an influx of the gammadelta+ subset, while CD4+ and CD8+ subsets did not accumulate in the pleural cavity after LPS stimulation. All those changes had resolved 96 h after LPS injection. Depletion of T lymphocytes by treatment with mAb anti-Thy 1.0 inhibited the eosinophil accumulation triggered by LPS. Aiming to clarify which T lymphocyte subset would be involved in the LPS-induced eosinophil accumulation, we depleted mice of various T lymphocyte subpopulations using specific Abs. Depletion of either CD4+ or CD8+ subsets failed to inhibit LPS-induced eosinophil migration. In contrast, when mice were treated with anti-gammadelta+ T lymphocyte mAb, a significant reduction of LPS-induced eosinophil accumulation was observed. Similarly, the administration of LPS in BALB/c-nu/nu mice induced the expected significant influx of eosinophils into the pleural cavity. Our results indicate that the gammadelta+ T lymphocytes are centrally involved in LPS-induced eosinophil accumulation in mice.     

Tumour necrosis factor-alpha expression and cell recruitment in Sephadex particle-induced lung inflammation: effects of dexamethasone and cyclosporin A

1. Tumour necrosis factor-alpha (TNF-alpha) is a cytokine with diverse properties consistent with a possible role in inflammatory disease. We investigated whether TNF-alpha is induced during the progression of lung inflammation elicited by a particulate non-antigenic stimulus, and whether pharmacological control of TNF-alpha expression influences recruitment of specific inflammatory cell types. 2. A single intravenous injection of Sephadex particles into rats led to extensive granulomatous inflammation in lung alveolar and bronchial tissue that peaked in intensity after 24-72 h. Mononuclear cells were the principal component of granulomas, but neutrophils and eosinophils were also abundant. Numbers of mononuclear cells, neutrophils and eosinophils recovered by bronchoalveolar lavage (BAL) peaked at 72 h, 48 h and 72 h, respectively. 3. Messenger RNA encoding TNF-alpha was induced in lung epithelial cells, lung granulomas and BAL cells 6 h after Sephadex administration and remained elevated for 72 h before declining to baseline by 7 days. In BAL cell populations TNF-alpha protein was localized to mononuclear cells at all times points pre- and post-Sephadex administration. 4. Treatment of rats with dexamethasone significantly reduced the Sephadex-induced recruitment of mononuclear cells, neutrophils and eosinophils into the bronchoalveolar cavity, and significantly reduced TNF-alpha mRNA expression by BAL cells. 5. Treatment of rats with cyclosporin A was without effect on Sephadex-induced elevations of mononuclear cell numbers and expression of TNF-alpha, but did reduce significantly recruitment of neutrophils and eosinophils to BAL cell populations. 6. These results show that a sequential asthma-like recruitment of neutrophils, eosinophils and mononuclear cells into lung tissue can be induced by single exposure to a non-antigenic stimulus. Pharmacological and histological studies reveal that mononuclear cell mobilization relates closely to induced TNF-alpha expression, whereas mobilization of neutrophils and eosinophils appears secondary to expression of the cytokine.     

Decision-making about genetic testing among women at familial risk for breast cancer

PURPOSE: To explore the effects on the lacrimal gland of adenovirus infection of the cornea. METHODS: Rabbit corneas were inoculated with human adenoviruses Ad5, Ad14, or a rabbit adapted form of Ad 5, and in some instances booster inoculations were given. Sections of lacrimal glands removed 21-59 days post-inoculation were immunostained using antibodies against rabbit Class I and Class II MHC molecules, CD4, CD8, CD18, and rabbit thymic lymphocyte antigen (RTLA). Relative numbers of positively stained cells were quantified with a Metamorph image analysis system. RESULTS: RTLA and CD18 antigens were expressed on many interstitial cells in the normal lacrimal gland, but few expressed CD4 or CD8. The number of RTLA+ cells increased by 60-100% after inoculation of Ad5 and after boosting, and CD18+ cells increased from 33-100% after inoculation of Ad5 and after boosting. Booster inoculations also caused focal lymphocytic infiltration. MHC Class I was expressed on interstitial cells and duct epithelium, but not acinar cells, and there was no detectable difference after viral infection. In controls, MHC Class II was localized to a population of interstitial cells and a few acinar cells. A single inoculation of the Ad5 virus did not result in an increase in the total number of MHC Class II-positive cells at 21 days, but inoculation with the rabbit-adapted Ad 5 and booster inoculations caused a 30% increase. CONCLUSIONS: Ad5 and rabbit-adapted Ad5 infection of the cornea induce lymphocytic infiltration in the lacrimal gland, and the effect is enhanced by boosting. There is also an increase in expression of MHC Class II after inoculation with rabbit-adapted Ad5 and with booster inoculations.     

Effects of thalidomide on neutrophil respiratory burst, chemotaxis, and transmigration of cytokine- and endotoxin-activated endothelium

Vascular endothelium activated by endotoxin and cytokines plays an important role in organ inflammation and blood leukocyte recruitment. Neutrophils, which are a homogeneous population of effector cells, are rapidly attracted in large numbers to sites of inflammation where they form an early response to infection or injury. Excessive production of various interleukins, TNF, arachidonic acid metabolites, and other substances by neutrophils and macrophages results in systemic endothelial cell injury, a fundamental problem. In the present study, we investigated in vitro the effects of thalidomide (THD) on activation of endothelial cells for enhanced transmigration of neutrophils by lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF), and interleukin-1 (IL-1). Modulation of endotoxin- and cytokine-induced neutrophil chemotaxis and respiratory burst by THD were also studied. Treatment of HUVEC with THD in combination with LPS, TNF, and IL-1, respectively, antagonized LPS-activated transmigration of neutrophils but stimulated the effects of TNF and IL-1. All of the agents used-THD, LPS, TNF, and IL-1-inhibited neutrophil chemotaxis. Addition of THD to the neutrophils had no effect on LPS-inhibited chemotaxis whereas the TNF- and IL-1-induced chemotaxis was modulated in a bimodal manner. However, THD failed to influence neutrophil respiratory burst activity. Results demonstrate that THD differentially affects mediator-induced activation of HUVEC and neutrophils.     

The role of LDL in the origin and progression of atherosclerosis: pathobiological concepts on the origin and development of atherosclerotic lesions and the role of the endothelium

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.     

Differences in eosinophil migration into the lungs following inhaled antigen challenge of parasite-naive guinea pigs with genetically determined differences in responsiveness to a gastrointestinal nematode parasite

Parasite-naive guinea pigs with genetically determined differences in responsiveness to infection with the gastrointestinal nematode parasite Trichostrongylus colubriformis were sensitised to ovalbumin and later challenged by exposure to an ovalbumin aerosol. The resultant cellular migration into the lungs was assessed by histological examination of the lungs and enumeration of cells in bronchoalveolar lavage fluid 24 h, 72 h and 7 days later. Compared with parasite-low-responder guinea pigs, there were approximately 10 times more eosinophils in lavage fluid from parasite-high-responder animals but similar numbers of neutrophils.     

Physical and cytochemical properties of neutrophils activated in situ in the lung during ZAP infusion in sheep

Increased retention of activated neutrophils in the lungs contributes to endothelial cell injury. However, characterization of the morphological changes that occur in neutrophils during activation in the pulmonary microcirculation has not been fully determined in vivo. Therefore, the present study was designed to determine structural and cytochemical properties of neutrophils in situ in pulmonary arterioles and alveolar capillaries during the infusion of zymosan-activated plasma (ZAP) or plasma (control) in anesthetized sheep. Quantitative morphological methods showed that ZAP infusion caused significant retention of neutrophils in alveolar capillaries [2.19 +/- 0.40 (SD) x 10(8) neutrophils/ml of capillary blood volume] and pulmonary arterioles (1.02 +/- 0.46 x 10(8) neutrophils/ml of arterial blood volume) compared with plasma infusion (1.03 +/- 0.15 and 0.30 +/- 0.10 x 10(8) neutrophils/ml, respectively; P < 0.05). Harmonic mean diameter of ZAP-activated neutrophils in situ (7.19 +/- 0.44 microns) was significantly greater than the diameter of neutrophils in plasma-treated sheep (6.29 +/- 0.17 microns; P < 0.05). Neutrophil cross-sectional area (54 +/- 3 microns2) and volume (248 +/- 27 microns3) in situ in alveolar capillaries were also significantly greater in ZAP-treated sheep than in control sheep (41 +/- 4 microns2 and 184 +/- 9 microns3, respectively; P < 0.05). Similarly, microvascular neutrophils in ZAP-treated sheep were vacuolated and elongated, filamentous actin was redistributed peripherally, and the cells were degranulated. We conclude that during ZAP infusion, neutrophils become enlarged and degranulated in pulmonary microvessels, especially in alveolar capillaries. The structural and cytochemical changes that occur are consistent with the hypothesis that neutrophil activation is accompanied by alterations in neutrophil physical properties, alterations that may facilitate retention and contribute to endothelial cell injury.