Bronchoalveolar Macrophages Research Articles

Antifungal mechanisms of activated murine bronchoalveolar or peritoneal macrophages for Histoplasma capsulatum.

The first line of defence against natural infection by Histoplasma capsulatum (Hc) consists of bronchoalveolar macrophages (BAM) and an early inflammatory response in the lungs. Little is known about the interaction of BAM and Hc, consequently we studied murine BAM in vitro to assess their role in the pulmonary defence in histoplasmosis. A short-term 3-h assay was used to measure fungicidal activity of control BAM and interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS)-activated BAM. Fungistatic activity of BAM was determined with a 24-h assay. A method devised for measuring colony-forming units (CFU) of non-ingested non-adherent and adherent ingested yeast cells of Hc in BAM cocultures was used. Activated BAM killed Hc (reduced inoculum CFU by 25 +/- 12%; n = 4). The fungicidal activity of BAM was abrogated by 0.2 mM NG-monomethyl-L-arginine (NMMA) or catalase but not by superoxide dismutase. In fungistatic assays activated BAM inhibited multiplication of Hc by 61 +/- 4% (n = 3) compared with cocultures with control BAM. However, Hc multiplied 100% more in control BAM cocultures than in medium alone. Data indicated that this was due to advantages that Hc has in the intracellular environment. Only NMMA inhibited fungistatic activity of activated BAM. In experiments with peritoneal macrophages (PM), results similar to those with BAM were obtained. In conclusion, activated BAM and PM kill yeast cells of Hc by a mechanism dependent on hydrogen peroxide and products of the nitric oxide synthase (NOS) pathway, whereas fungistasis depends only on products of the NOS pathway.

Normal and sarcoid alveolar macrophages differ in their ability to present antigen and to cluster with autologous lymphocytes

Human bronchoalveolar macrophages from normal individuals function poorly as accessory cells for the presentation of common recall antigens. In sarcoidosis, alveolar macrophages (AM) are reported to be effective accessory cells for the presentation of such antigens. In this study normal and sarcoid AM were compared with blood monocytes for their ability to act as accessory cells in presenting tuberculin purified protein derivative (PPD) and streptokinase-streptodornase (SKSD) to autologous T lymphocytes, or to form spontaneous, antigen- or mitogen-induced clusters with the T cells. When compared to autologous monocytes, normal AM failed to present the two recall antigens effectively. Likewise normal AM formed very few clusters with T lymphocytes when compared to monocytes, even in the presence of antigens or the mitogen phytohaemagglutinin (PHA). In contrast, sarcoid AM presented both antigens as effectively, and were equally effective as monocytes in forming clusters with T lymphocytes, spontaneously and in further response to antigen or mitogen. The results suggest that in sarcoidosis enhanced accessory cell function and enhanced cluster formation may be related features of bronchoalveolar macrophage populations.

Envelope glycoprotein (gp120) from HIV-1 enhances Mycobacterium avium growth in human bronchoalveolar macrophages; an effect mediated by enhanced prostaglandin synthesis.

Human bronchoalveolar lavage (BAL) macrophages were obtained from normal human volunteers and infected with an AIDS-associated strain of Mycobacterium avium. Infected cells were exposed to purified envelope glycoprotein (gp120) from HIV-1 or to the recombinant non-glycosylated gp120 fragments PBI-RF and PBI-IIIB. Native gp120 increased Myco. avium growth in human cells from six separate donors, whereas the non-glycosylated fragments of gp120 had no such effect. Moreover, gp120 induced a substantial secretion of prostaglandin E2 (PGE2) from macrophages; inclusion of indomethacin blocked the enhanced permissiveness of infected cells treated with gp120. Soluble CD4 also neutralized the effect of gp120. Overall, these results indicate a role for gp120 in the susceptibility of AIDS patients to Myco. avium infections, mediated by an enhanced PGE2 release.

Development and validation of an oligonucleotide microarray for immuno-inflammatory genes of ruminants

This report describes the development of small DNA microarrays of fully defined genes suitable for projects requiring detailed analysis of gene expression in sheep and/or cattle. Two arrays have been developed; the first is a small reference microarray (RIGRA) that has been used to validate experimental design and methodology; the second, a larger array (RIGUA) containing probes for 516 ruminant immuno-inflammatory genes, each represented by non-overlapping 75mer oligonucleotides. Experiments used to validate this microarray were: (1) a comparison of gene expression profiles from sheep broncho-alveolar macrophages before and after in vitro activation with lipopolysaccharide (LPS), using the RIGRA; (2) the differential gene expression between five in vitro unstimulated sheep keratinocyte cultures; (3) LPS/interferon gamma stimulated and unstimulated blood monocytes purified from Holstein-Friesians (Bos taurus) and Sahiwals (Bos indicus) cattle using the RIGUA. Real-time, quantitative RT-PCR was used to validate the gene expression profiles obtained with the RIGUA microarrays. The potential for using such an immunological tool in understanding the relative gene expression corresponding to immune-inflammatory responses of sheep and cattle is discussed.

Modulation of ozone-induced airway hyperresponsiveness and inflammation by interleukin-13

The present study aimed to determine whether the T-helper cell type 2-derived cytokines, interleukin (IL)-4 and -13, can modulate the lung response to ozone exposure. IL-13(-/-), IL-4/13(-/-) and IL-13-overexpressing transgenic (Tg) mice were exposed to ozone (3 ppm; 3 h) or air. Wild-type (Wt) Balb/c mice and transgenic-negative littermates (IL-13Wt) were used as controls for gene-deficient and IL-13Tg mice, respectively. IL-4/13(-/-) and IL-13(-/-) mice developed a lesser degree of ozone-induced airway hyperresponsiveness (AHR) while IL-13Tg mice developed a greater degree of AHR compared with ozone-exposed wild-type or IL-13Wt mice, respectively. Ozone caused a time-dependent increase of bronchoalveolar lavage (BAL) neutrophils and macrophages in wild-type mice, maximal at 20-24 h, which was attenuated in the IL-13(-/-) and IL-4/13(-/-) mice. In IL-13Tg mice, there was a greater increase in BAL neutrophils after ozone exposure compared with IL-13Wt mice. Using quantitative real-time PCR, ozone-induced mRNA expression for IL-6 and keratinocyte chemokine was further enhanced in IL-13(-/-) and IL-4/13(-/-) mice, and was inhibited in IL-13Tg mice. Macrophage inflammatory protein (MIP)-3alpha/CCL20 expression was enhanced after ozone exposure in wild-type mice, inhibited in IL-13(-/-) and IL-4/13(-/-) mice, while in IL-13Tg mice it was enhanced. A similar pattern of expression was observed with lipopolysaccharide-induced cytokine (LIX/CXCL5/ENA-78) expression. In conclusion, interleukin-13 augments ozone-induced airway hyperresponsiveness and neutrophilic inflammation, possibly through modulation of certain cytokines induced by ozone exposure.

Inflammation and ischemia-induced lung angiogenesis

A role for inflammation in modulating the extent of angiogenesis has been shown for a number of organs. The present study was undertaken to evaluate the importance of leukocyte subpopulations for systemic angiogenesis of the lung after left pulmonary artery ligation (LPAL) in a mouse model of chronic pulmonary thromboembolism. Since we (24) previously showed that depletion of neutrophils did not alter the angiogenic outcome, we focused on the effects of dexamethasone pretreatment (general anti-inflammatory) and gadolinium chloride treatment (macrophage inactivator) and studied Rag-1(-/-) mice (T/B lymphocyte deficient). We measured inflammatory cells in bronchoalveolar lavage fluid and lung homogenate macrophage inflammatory protein-2 (MIP-2) and IL-6 protein levels within 24 h after LPAL and systemic blood flow to the lung 14 days after LPAL with labeled microspheres as a measure of angiogenesis. Blood flow to the left lung was significantly reduced after dexamethasone treatment compared with untreated control LPAL mice (66% decrease; P < 0.05) and significantly increased in T/B lymphocyte-deficient mice (88% increase; P < 0.05). Adoptive transfer of splenocytes (T/B lymphocytes) significantly reversed the degree of angiogenesis observed in the Rag-1(-/-) mice back to the level of control LPAL. Average number of lavaged macrophages for each group significantly correlated with average blood flow in the study groups (r(2) = 0.9181; P = 0.01 different from 0). Despite differences in angiogenesis, left lung homogenate MIP-2 and IL-6 did not differ among study groups. We conclude that inflammatory cells modulate the degree of angiogenesis in this lung model where lymphocytes appear to limit the degree of neovascularization, whereas monocytes/macrophages likely promote angiogenesis.

Coagulation Factor Xa Modulates Airway Remodeling in a Murine Model of Asthma

Previous studies have demonstrated that dysregulated coagulation and fibrinolysis contribute to the pathogenesis of asthma. The role of procoagulant factor X in a murine model of ovalbumin (OVA)-induced asthma was investigated. Biochemical, cellular, and physiologic in vivo and in vitro approaches were used to determine effects of factor X on the asthmatic response in mice. Factor X transcript levels and factor Xa activity were increased in lungs of asthmatic mice challenged with OVA, compared with controls treated with phosphate-buffered saline. Factor X was highly expressed in bronchoalveolar lavage fluid macrophages from asthmatic mice. Treatment of mice with the factor Xa inhibitor fondaparinux during the last 4 wk of OVA challenge resulted in the attenuation of airway hyperresponsiveness but did not alter infiltration of inflammatory cells into the lung. There was a significant decrease in the thickness of the mucosal layer and in lung collagen deposition in fondaparinux-treated mice. In vitro investigations using human mucus-producing NCI-H292 cells indicated that exogenous factor Xa enhanced mucin production in a dose-dependent manner. Levels of amphiregulin, a protein that induces mucin production, were also increased in cells stimulated by factor Xa. The results of this study introduce a novel participant in the asthmatic response and indicate that factor Xa functions in airway remodeling in asthma by stimulating mucin production, through regulation of amphiregulin expression and collagen deposition.

Human rhinovirus induces robust IP-10 release by monocytic cells, which is independent of viral replication but linked to type I interferon receptor ligation and STAT1 activation

Human rhinovirus (HRV)-induced respiratory infections are associated with elevated levels of IFN-gamma-inducible protein 10 (IP-10), which is an enhancer of T lymphocyte chemotaxis and correlates with symptom severity and T lymphocyte number. Increased IP-10 expression is exhibited by airway epithelial cells following ex vivo HRV challenge and requires intracellular viral replication; however, there are conflicting reports regarding the necessity of type I IFN receptor ligation for IP-10 expression. Furthermore, the involvement of resident airway immune cells, predominantly bronchoalveolar macrophages, in contributing to HRV-stimulated IP-10 elaboration remains unclear. In this regard, our findings demonstrate that ex vivo exposure of human peripheral blood monocytes and bronchoalveolar macrophages (monocytic cells) to native or replication-defective HRV serotype 16 (HRV16) resulted in similarly robust levels of IP-10 release, which occurred in a time- and dose-dependent manner. Furthermore, HRV16 induced a significant increase in type I IFN (IFN-alpha) release and STAT1 phosphorylation in monocytes. Neutralization of the type I IFN receptor and inhibition of JAK or p38 kinase activity strongly attenuated HRV16-stimulated STAT1 phosphorylation and IP-10 release. Thus, this work supports a model, wherein HRV16-induced IP-10 release by monocytic cells is modulated via autocrine/paracrine action of type I IFNs and subsequent JAK/STAT pathway activity. Our findings demonstrating robust activation of monocytic cells in response to native and/or replication-defective HRV16 challenge represent the first evidence indicating a mechanistic disparity in the activation of macrophages when compared with epithelial cells and suggest that macrophages likely contribute to cytokine elaboration following HRV challenge in vivo.

IL-12 induction of resistance to pulmonary blastomycosis

Background Why severity varies in blastomycosis outbreaks remains unresolved. In experimental pulmonary blastomycosis, susceptibility varied in mouse strains. In susceptible BALB/c the response is Th-2, in immunized, resistance is associated with Th-1. Can susceptibility be redirected by IL-12? Methods, results: BALB/c bronchoalveolar and peritoneal macrophages (PM) were shown deficient in IL-12 production in response to IFN-γ + LPS. High dose IL-12 (1 μg, subcutaneously) treatment of BALB/c infected intranasally with Blastomyces resulted in enhanced survival ( P < 0.008). Since IL-12 was poorly tolerated, a new protocol for infected mice, IL-12 0.1 or 0.3 μg, every other day, resulted in minimal toxicity; almost all treated mice survived ( P < 0.002 vs. controls). When lungs of surviving mice were cultured, the 0.1 μg regimen resulted in fewer ( P < 0.02) cfu. For weeks after treatment, in vitro IFN-γ treatment enabled PM Blastomyces killing. After infection spleen cells from IL-12 treated mice produced 4-fold more IFN-γ and 3-fold less IL-10 in response to Blastomyces. IL-10 abrogated activation of macrophages by IFN-γ for enhanced Blastomyces killing. Conclusions: A proper IL-12 treatment protocol induces resistance (survival and decreased growth in lungs), low toxicity, macrophage responsiveness to IFN-γ for killing Blastomyces, up-regulation of IFN-γ and down-regulation of IL-10 production.

Effect of Lung Surfactant Collectins on Bronchoalveolar Macrophage Interaction withBlastomyces dermatitidis: Inhibition of Tumor Necrosis Factor Alpha Production by Surfactant Protein D

Alveolar surfactant modulates the antimicrobial function of bronchoalveolar macrophages (BAM). Little is known about the effect of surfactant-associated proteins in bronchoalveolar lavage fluid (BALF) on the interaction of BAM and Blastomyces dermatitidis. We investigated BALF enhancement or inhibition of TNF-alpha production by BAM stimulated by B. dermatitidis. BAM from CD-1 mice were stimulated with B. dermatitidis without or with normal BALF, surfactant protein A-deficient (SP-A-/-) or surfactant protein D-deficient (SP-D-/-) BALF, or a mixture of SP-A-/- and SP-D-/- BALF. An enzyme-linked immunosorbent assay was used to measure tumor necrosis factor alpha (TNF-alpha) in culture supernatants. BALFs were standardized in protein concentration. BAM plus B. dermatitidis (BAM-B. dermatitidis) TNF-alpha production was inhibited > or = 47% by BALF or SP-A-/- BALF (at 290 or 580 microg of protein/ml, P < 0.05 to 0.01); in contrast, SP-D-/- BALF did not significantly inhibit TNF-alpha production. If SP-A-/- BALF was mixed in equal amounts with SP-D-/- BALF, TNF-alpha production by BAM-B. dermatitidis was inhibited (P < 0.01). Finally, pure SP-D added to SP-D-/- BALF inhibited TNF-alpha production by BAM-B. dermatitidis (P < 0.01). B. dermatitidis incubated with BALF and washed, plus BAM, stimulated 63% less production of TNF-alpha than did unwashed B. dermatitidis (P < 0.05). SP-D was detected by anti-SP-D antibody on BALF-treated unwashed B. dermatitidis in an immunofluorescence assay (IFA). The BALF depleted by a coating of B. dermatitidis lost the ability to inhibit TNF-alpha production (P < 0.05). 1,3-beta-Glucan was a good stimulator of BAM for TNF-alpha production and was detected on B. dermatitidis by IFA. beta-Glucan incubated with BALF inhibited the binding of SP-D in BALF to B. dermatitidis as demonstrated by IFA. Our data suggest that SP-D in BALF binds beta-glucan on B. dermatitidis, blocking BAM access to beta-glucan, thereby inhibiting TNF-alpha production. Thus, whereas BALF constituents commonly mediate antimicrobial activity, B. dermatitidis may utilize BALF constituents, such as SP-D, to blunt the host defensive reaction; this effect could reduce inflammation and tissue destruction but could also promote disease.

Regulation of Chemokine Expression by NaCl Occurs Independently of Cystic Fibrosis Transmembrane Conductance Regulator in Macrophages

Chronic pulmonary inflammation and infection are the leading causes of morbidity and mortality in cystic fibrosis (CF). While the effect of mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) on airways remains controversial, some groups have demonstrated increases in Na(+) and Cl(-) in CF airway surface liquid compared to normal airways. We investigated the consequences of NaCl on pro-inflammatory chemokine and cytokine production by macrophages. Stimulation of mouse macrophages with increasing amounts of NaCl induced macrophage inflammatory protein-2 (MIP-2) and tumor necrosis factor-alpha (TNF-alpha) production. Further, co-incubation of macrophages with NaCl in the presence of either lipopolysaccharide (LPS) or TNF-alpha synergistically increased MIP-2 production. Both the NaCl and NaCl plus LPS responses were partially dependent on endogenous production and autocrine signaling by TNF-alpha. To investigate the role of CFTR in MIP-2 production, we compared the responses of wild-type and DeltaF508 CF mouse macrophages to NaCl and LPS. The responses of macrophages from both strains were indistinguishable. In addition, CFTR mRNA was not expressed in macrophages. Taken together, these findings suggest that NaCl stimulates MIP-2 production by macrophages through a mechanism that is partially dependent on TNF-alpha but independent of macrophage CFTR expression.

Overexpression of Bcl-2 Protein in Bronchoalveolar Lavage Lymphocytes and Macrophages in Sarcoidosis

Background and Objectives: Apoptosis, also known as programmed cell death, probably correlates with the pathophysiologic mechanisms of alveolitis in sarcoidosis. Our purpose was to investigate any changes in the expression of the antiapoptotic protein Bcl-2, one of the most important inhibiting factors of apoptosis, in bronchoalveolar lavage fluid (BALF) cell populations in patients with sarcoidosis. Subjects and Methods: Fiberoptic bronchoscopy with BAL was performed in 13 patients with active sarcoidosis (10 patients with stage I and 3 with stage II disease based on chest radiography). None of them was under treatment with corticosteroids. Cellular Bcl-2 expression was identified using an immunoperoxidase staining method. Ten normal subjects served as control group. Results: BALF lymphocytes and macrophages in sarcoid patients exhibited a significant increase in the expression of Bcl-2 compared with the control group (p < 0.001). A Bcl-2 expression of 80.7 ± 8.5% in the lymphocytes and 77.4 ± 8.9% in the macrophages was observed in sarcoidosis versus 32.2 ± 13.8% and 19.6% ± 7.6% in normal subjects, respectively. The percentages of Bcl-2 expression in the lymphocytes were positively correlated with BALF CD4/CD8 ratio values (p = 0.02, r = 0.63). Conclusions: Our results suggest that the antiapoptotic protein Bcl-2 is overexpressed in alveolar lymphocytes and macrophages, which characterize the alveolitis in sarcoidosis and could provide insights into the pathogenesis of the disease and prove useful as a marker of disease activity or response to therapy.

Susceptibility to pulmonary blastomycosis in young compared to adult mice: immune deficiencies in young mice

The immunological basis for differences in resistance to pulmonary blastomycosis between young (3 to 4-week-old) and adult (7 to 8-week-old) CD-1 mice is unknown. We assessed whether there were differences in fungicidal activity of phagocytes and Th-1 lymphocyte cytokine production. The fungicidal activity of young bronchoalveolar macrophages (BAM) (20%) against Blastomyces dermatitidis (Bd) was comparable to killing by adult BAM (25%). However, IFN-gamma enhanced the killing by adult BAM (from 30 to 69%) to a greater extent than BAM from young animals (from 20 to 30%). Killing of Bd by young peritoneal macrophages (PM) (46%) and adult PM (42%) was similar, and the enhancement of cells of both by IFN-gamma was similar. TNFalpha production by young macrophages (BAM or PM), when cocultured with Bd for 18 h, was half of TNFalpha secreted by adult macrophages. We found that polymorphonuclear neutrophils (PMN) from young mice had deficient fungicidal activity against Bd (37%) compared with adult PMN (80%). Interferon-gamma (IFN-gamma) treatment increased PMN killing of Bd by PMN of young animals from 37 to 80%. In an assessment of innate responses, we found spleen cells from young mice produced three-fold less IFN-gamma and three-fold less IL-2 than adult spleen cells in response to 1 microg/ml concanavalin A (Con A). The young spleen cells also produced more NO, which we demonstrated reduced Con A-induced proliferation. These in vitro results demonstrate several immunological deficiencies in cells from young mice and these deficiencies correlate with susceptibility. In a pilot reconstitution experiment in pulmonary blastomycosis, treatment of infected young mice with IFN-gamma (18.5 x 10(3) U, s.c.) on days 0, 1, and 2 significantly increased survival.

Effects of cyclic AMP-elevating hormones and autacoids on LPS-activated rat peritoneal, bronchoalveolar and hepatic (Kupffer) macrophages

Peritoneal, bronchoalveolar and hepatic (Kupffer) macrophages activated in vitro by endotoxin, exhibit alterations in nitric oxide production when certain hormones or other biologically active agents (autacoids) are present in the culture medium. They also show changes in acid beta-glucuronidase activities and morphological changes concerning cell size and general appearance. Agents known to elevate the intracellular levels of cyclic AMP, e.g. adrenalin, prostaglandin E2 and dopamine, increase the nitric oxide production in all three types of macrophage. The addition of H-89, an inhibitor of protein kinase A, abolishes the increase in nitric oxide production. Adrenalin also increases the extracellular activity of beta-glucuronidase. The results of this work suggest that cyclic AMP-elevating hormones and autacoids affect the functions of endotoxin-activated macrophages, such as the production of nitric oxide and the activity of acid beta-glucuronidase.

Gene expression profiles of U937 human macrophages exposed to Chlamydophila pneumoniae and/or low density lipoprotein in five study models using differential display and real-time RT-PCR

Many studies on atherosclerosis have indicated an association with Chlamydophila pneumoniae, a respiratory intracellular pathogen that can infect bronchoalveolar macrophages, and be transported to sites of vascular injury. Furthermore, the connection between low density lipoprotein (LDL), macrophages, foam cell formation, and atherosclerosis is well established. Five study models were designed to mimic the early events in plaque formation, i.e. untreated U937 human macrophages as control; macrophages treated with LDL only; macrophages infected with the AR-39 strain of C. pneumoniae only; and the last two models in which macrophages were exposed to both C. pneumoniae and LDL but in different order. Differential display RT-PCR was employed to analyze mRNAs from the five models to elucidate the transcriptional responses arising from macrophage interactions with C. pneumoniae and/or LDL. Out of 235 expressed sequence tags, the altered expression patterns of 29 selected genes at various time-points were authenticated by semi-quantitative and real-time RT-PCR. In relation to atherosclerosis, some interesting modified transcripts are involved in cell proliferation (CDC2, CDC27, CDC42EP3, GBDR1, GPR6, RPL27A, Similar to COX2), apoptosis (BCL2A1), ubiquitination (CTSH, HSPC150, NUC2), and structural integrity (ACTG1, FLOT1). Other genes exhibiting altered expression include those that modulate inflammatory and immune responses or signal transduction linking infection to host immunity (ARPP-19, BLAME, HCA58, IK cytokine, IRS2, STAT1P1). The transcriptional alterations of critical human genes in response to C. pneumoniae infection and foam cell development provide insights into certain key molecular pathophysiological mechanisms of atherosclerosis.

Immunological basis for susceptibility and resistance to pulmonary blastomycosis in mouse strains

The immunological basis for a >10-fold resistance of outbred CD-1 mice compared to inbred BALB/c mice to pulmonary blastomycosis was investigated. Bronchoalveolar macrophages (BAM) from CD-1 mice killed yeast cells of Blastomyces dermatitidis (Bd) by 25% and this increased to 59% when activated by IFN-γ. In contrast, BAM from BALB/c mice lacked significant killing (5%) of Bd but could be activated by IFN-γ for enhanced killing (19%). Peritoneal macrophages (PM) from CD-1 mice had significant fungicidal activity for Bd (43%) and this increased to 63% with IFN-γ treatment. By contrast, PM from BALB/c mice did not significantly kill Bd (14%) but were activated by IFN-γ for significant killing (24%). Fungicidal activity of peripheral blood polymorphonuclear neutrophils (PMN) from CD-1 (87%) was greater than that of BALB/c (75%) ( P < 0.05). Macrophage inflammatory protein-1α (MIP-1α) production by BAM from BALB/c was significantly less than that from CD-1 in response to co-culture with Bd. IFN-γ production by CD-1 spleen cells in response to concanavalin A (Con A, 1 μg/ml) was 8-fold greater than that by BALB/c spleen cells. In contrast, BAM and PM from BALB/c mice in co-culture with Bd secreted several-fold more TNFα than BAM or PM from CD-1 mice. IL-2 production by BALB/c spleen cells in response to Con A was 3- to 4-fold greater than that by CD-1 spleen cells. Depressed IL-2 production by Con A stimulated CD-1 spleen cells correlated with depressed proliferative responses. Resistance of CD-1 mice to pulmonary blastomycosis correlates with enhanced fungicidal activity of BAM, PM, PMN, and IFN-γ production by Con A stimulated spleen cells, compared to BALB/c mice. Consistent with the in vitro enhancement of effector cell function by IFN-γ, in vivo therapy with IFN-γ significantly ( P < 0.0001) improved survival of BALB/c mice with pulmonary blastomycosis.

Microparticle-based lung delivery of INH decreases INH metabolism and targets alveolar macrophages

Microparticles prepared by the precipitation with a compressed antisolvent (PCA) process were evaluated for their potential in targeting an ionizable prodrug of isoniazid (INH), isoniazid methanesulfonate (INHMS), for sustained delivery of INH to alveolar macrophages (AMs). The charged prodrug was ion-paired with two different hydrophobic cations (tetrapentylammonium (TPA)— and tetraheptylammonium (THA)—bromide), and loaded separately into the poly( l-lactide) (PLA) microparticles. The drug/polymer particles were spherical in shape and between 1 and 3 μm in diameter. The choice of hydrophobic cations did not affect drug incorporation efficiencies or the release kinetics of INH from the microparticles. Using a sensitive liquid chromatographic tandem mass spectrometric (LC-MS/MS) assay developed for INH, high level of INH was detected in NR8383, a rat AM cell line, following exposure of these cells to drug-loaded microparticles. To confirm the microparticles can target AMs in vivo, we compared the INH levels in lavaged bronchoalveolar macrophages by LC-MS/MS after the Sprague–Dawley rats were administered either INHMS in PLA microparticles by intra-tracheal instillation or INH solution by gavage or intra-tracheal instillation. As expected, only microparticles provided sustained and targeted delivery of INH to AMs. Most importantly, this method of delivery led to substantial reduction in the blood levels of acetylisoniazid (AcINH), a major and potential toxic metabolite of INH.

The influence of acute hypercapnia and priming of 10 ng/kg endotoxin on oxidative metabolism of bronchoalveolar lavage-derived alveolar macrophages in mechanically ventilated rabbits

Misztal, B.; Makowski, A.; Billert, H.; Safranow, K.; Marzec-Lewenstein, E.; Bohatyrewicz, R. Author Information

An increase in exhaled nitric oxide is not associated with activity in pulmonary sarcoidosis.

Exhaled nitric oxide (eNO) concentration measurement may permit the noninvasive estimation of the severity of airways inflammation in respiratory tract diseases. The aim of this study was to evaluate the correlation between eNO concentration and the activity of sarcoidosis, its radiographic staging and lung function abnormalities. eNO concentration was measured using a chemiluminescent analyser in 27 patients with sarcoidosis and 11 control subjects. The mean eNO concentration in patients with sarcoidosis was significantly higher (6.7+/-0.50 parts per billion (ppb)) than that in the control group (5.17+/-0.73 ppb). eNO concentration was similar in radiographic stage I, II and III patients (6.53, 7.32 and 6.24 ppb, respectively). No significant difference was found in eNO concentration between the patients with active and inactive disease. Nor did eNO concentration differ between the patients with and without indication for therapy. There was no significant correlation between eNO concentration and forced vital capacity or bronchoalveolar lavage fluid lymphocyte and macrophage counts. There was a weak correlation between eNO concentration and single-breath carbon monoxide diffusing capacity of the lung. Exhaled nitric oxide concentration is elevated in patients with sarcoidosis. This concentration does not depend on the radiographic staging, activity or progression of the disease.

IPL576,092, a novel anti-inflammatory compound, inhibits leukocyte infiltration and changes in lung function in response to allergen challenge

IPL576,092, a lead compound from a novel class of polyhydroxylated sterols, was tested in models of allergen-induced bronchoconstriction and airway inflammation. In a rat ovalbumin lung inflammation model, orally administered IPL576,092 significantly inhibited the challenge-mediated increase in total bronchoalveolar lavage leukocyte numbers, and macrophage and lymphocyte infiltration (1–10 mg/kg/day). There was a similar trend towards inhibition of eosinophil and neutrophil accumulation. Sheep were treated with IPL576,092 by inhalation (400 μg/kg/day), and lung resistance and airway hyper-responsiveness (AHR) were determined after Ascaris suum challenge. IPL576,092 significantly reduced the early and late phase bronchoconstrictor responses by 63±4.6 and 84±4.6%, respectively. IPL576,092 also blocked AHR (2.2±5.7% change from pre-challenge PC400), whereas control animals showed a 62.2±2.6% decrease in the PC400 ( p<0.05). Oral IPL576,092 (5 mg/kg/day) also significantly decreased hyper-reactivity in mice. In a guinea pig model, IPL576,092 (5 mg/kg/day) significantly protected against allergen-induced increases in lung resistance (11.4±2.3 control versus 4.8±01.5 IPL576,092, area under the curve) and inhibited the increase in lung elastance (280±58 control versus 167±52 IPL576,092, p<0.05). IPL576,092, unlike dexamethasone, did not significantly decrease rat serum corticosterone levels or thymus and spleen weights, supporting a mechanism of action different from classic glucocorticoids. IPL576,092 significantly attenuates characteristics of an asthmatic response, indicating therapeutic potential for this drug class.