Lung-restricted macrophage activation in the pearl mouse model of Hermansky-Pudlak syndrome

Pulmonary inflammation, abnormalities in alveolar type II cell and macrophage morphology, and pulmonary fibrosis are features of Hermansky-Pudlak Syndrome (HPS). We used the naturally occurring "pearl" HPS2 mouse model to investigate the mechanisms of lung inflammation observed in HPS. Although baseline bronchoalveolar lavage (BAL) cell counts and differentials were similar in pearl and strain-matched wild-type (WT) mice, elevated levels of proinflammatory (MIP1gamma) and counterregulatory (IL-12p40, soluble TNFr1/2) factors, but not TNF-alpha, were detected in BAL from pearl mice. After intranasal LPS challenge, BAL levels of TNF-alpha, MIP1alpha, KC, and MCP-1 were 2- to 3-fold greater in pearl than WT mice. At baseline, cultured pearl alveolar macrophages (AMs) had markedly increased production of inflammatory cytokines. Furthermore, pearl AMs had exaggerated TNF-alpha responses to TLR4, TLR2, and TLR3 ligands, as well as increased IFN-gamma/LPS-induced NO production. After 24 h in culture, pearl AM LPS responses reverted to WT levels, and pearl AMs were appropriately refractory to continuous LPS exposure. In contrast, cultured pearl peritoneal macrophages and peripheral blood monocytes did not produce TNF-alpha at baseline and had LPS responses which were no different from WT controls. Exposure of WT AMs to heat- and protease-labile components of pearl BAL, but not WT BAL, resulted in robust TNF-alpha secretion. Similar abnormalities were identified in AMs and BAL from another HPS model, pale ear HPS1 mice. We conclude that the lungs of HPS mice exhibit hyperresponsiveness to LPS and constitutive and organ-specific macrophage activation.     

Phenotypic characterization of alveolar monocyte recruitment in acute respiratory distress syndrome

In 49 acute respiratory distress syndrome (ARDS) patients, the phenotype of alveolar macrophages (AMs) was analyzed by flow cytometry. Bronchoalveolar lavage (BAL) was performed within 24 h after intubation and on days 3-5, 9-12, and 18-21 of mechanical ventilation. The 27E10(high)/CD11b(high)/CD71(low)/ 25F9(low)/HLA DR(low)/RM3/1(low) AM population in the first BAL indicated extensive monocyte influx into the alveolar compartment. There was no evidence of increased local AM proliferation as assessed by nuclear Ki67 staining. Sequential BAL revealed two distinct patient groups. In one, a decrease in 27E10 and CD11b and an increase in CD71, 25F9, HLA DR, and RM3/1 suggested a reduction in monocyte influx and maturation of recruited cells into AMs, whereas the second group displayed sustained monocyte recruitment. In the first BAL from all patients, monocyte chemoattractant protein (MCP)-1 was increased, and AMs displayed elevated MCP-1 gene expression. In sequential BALs, a decrease in MCP-1 coincided with the disappearance of monocyte-like AMs, whereas persistent upregulation of MCP-1 paralleled ongoing monocyte influx. A highly significant correlation between BAL fluid MCP-1 concentration, the predominance of monocyte-like AMs, and the severity of respiratory failure was noted.     

Macrophage Activation in Acute Exacerbation of Idiopathic Pulmonary Fibrosis

Background

Acute exacerbation (AE) of idiopathic pulmonary fibrosis (IPF) is a common cause of disease acceleration in IPF and has a major impact on mortality. The role of macrophage activation in AE of IPF has never been addressed before.

Methods

We evaluated BAL cell cytokine profiles and BAL differential cell counts in 71 IPF patients w/wo AE and in 20 healthy volunteers. Twelve patients suffered from AE at initial diagnosis while sixteen patients developed AE in the 24 months of follow-up. The levels of IL-1ra, CCL2, CCL17, CCL18, CCL22, TNF-α, IL-1β, CXCL1 and IL-8 spontaneously produced by BAL-cells were analysed by ELISA.

Results

In patients with AE, the percentage of BAL neutrophils was significantly increased compared to stable patients. We found an increase in the production rate of the pro-inflammatory cytokines CXCL1 and IL-8 combined with an increase in all tested M2 cytokines by BAL-cells. An increase in CCL18 levels and neutrophil counts during AE was observed in BAL cells from patients from whom serial lavages were obtained. Furthermore, high baseline levels of CCL18 production by BAL cells were significantly predictive for the development of future AE.

Conclusions

BAL cell cytokine production levels at acute exacerbation show up-regulation of pro-inflammatory as well as anti-inflammatory/ M2 cytokines. Our data suggest that AE in IPF is not an incidental event but rather driven by cellular mechanisms including M2 macrophage activation.     

Immunopathology of a two-hit murine model of acid aspiration lung injury

In a two-hit model of acid aspiration lung injury, mice were subjected to nonlethal cecal ligation and puncture (CLP). After 48 h, intratracheal (IT) acid was administered, and mice were killed at several time points. Recruitment of neutrophils in response to acid was documented by myeloperoxidase assay and neutrophil counts in bronchoalveolar lavage (BAL) fluid and peaked at 8 h post-IT injection. Albumin in BAL fluid, an indicator of lung injury, also peaked at 8 h. When the contributions of the two hits were compared, neutrophil recruitment and lung injury occurred in response to acid but were not greatly influenced by addition of another hit. Neutrophil sequestration was preceded by elevations in KC and macrophage inflammatory protein-2alpha in plasma and BAL fluid. KC levels in BAL fluid were higher and peaked earlier than macrophage inflammatory protein-2alpha levels. When KC was blocked with specific antiserum, neutrophil recruitment was significantly reduced, whereas albumin in BAL fluid was not affected. In conclusion, murine KC mediated neutrophil recruitment but not lung injury in a two-hit model of aspiration lung injury.     

Serotype-dependent induction of pulmonary neutrophilia and inflammatory cytokine gene expression by reovirus.

Reovirus type 3 Dearing (T3D) causes a prominent neutrophil influx, substantially greater than seen with reovirus type 1 Lang (T1L) in a rat model of viral pneumonia. We sought to measure reovirus-mediated increases in chemokine mRNA expression in pulmonary cells. We found that the neutrophilia induced by T1L and T3D infection in vivo correlated directly with increased levels of chemokine mRNA expression in T3D-infected compared with those of T1IL-infected lungs. In vitro, reovirus-infected normal alveolar macrophages (AMs) and the rat AM cell line NR8383 expressed greater levels of macrophage inflammatory protein 2, KC, and tumor necrosis factor alpha mRNA. A synergism between reovirus and lipopolysaccharide was also detected for macrophage inflammatory protein 2 and KC mRNA expression. Tumor necrosis factor protein secretion was also increased to a greater extent by T3D than by T1L in primary rat AMs and the NR8383 cells. We conclude that the virus-mediated inflammatory cytokine induction suggests a role for these cytokines in the neutrophil influx observed in the rat reovirus pneumonia model.     

Ethanol induces oxidative stress in alveolar macrophages via upregulation of NADPH oxidases

Chronic alcohol abuse is a comorbid variable of acute respiratory distress syndrome. Previous studies showed that, in the lung, chronic alcohol consumption increased oxidative stress and impaired alveolar macrophage (AM) function. NADPH oxidases (Noxes) are the main source of reactive oxygen species in AMs. Therefore, we hypothesized that chronic alcohol consumption increases AM oxidant stress through modulation of Nox1, Nox2, and Nox4 expression. AMs were isolated from male C57BL/6J mice, aged 8-10 wk, which were treated with or without ethanol in drinking water (20% w/v, 12 wk). MH-S cells, a mouse AM cell line, were treated with or without ethanol (0.08%, 3 d) for in vitro studies. Selected cells were treated with apocynin (300 μM), a Nox1 and Nox2 complex formation inhibitor, or were transfected with Nox small interfering RNAs (20-35 nM), before ethanol exposure. Human AMs were isolated from alcoholic and control patients' bronchoalveolar lavage fluid. Nox mRNA levels (quantitative RT-PCR), protein levels (Western blot and immunostaining), oxidative stress (2',7'-dichlorofluorescein-diacetate and Amplex Red analysis), and phagocytosis (Staphylococcus aureus internalization) were measured. Chronic alcohol increased Nox expression and oxidative stress in mouse AMs in vivo and in vitro. Experiments using apocynin and Nox small interfering RNAs demonstrated that ethanol-induced Nox4 expression, oxidative stress, and AM dysfunction were modulated through Nox1 and Nox2 upregulation. Further, Nox1, Nox2, and Nox4 protein levels were augmented in human AMs from alcoholic patients compared with control subjects. Ethanol induces AM oxidative stress initially through upregulation of Nox1 and Nox2 with downstream Nox4 upregulation and subsequent impairment of AM function.     

Interactions of lung stretch, hyperoxia, and MIP-2 production in ventilator-induced lung injury.

The use of positive pressure mechanical ventilation can cause ventilator-induced lung injury (VILI). We hypothesized that hyperoxia in combination with large tidal volumes (VT) would accentuate noncardiogenic edema and neutrophil infiltration in VILI and be dependent on stretch-induced macrophage inflammatory protein-2 (MIP-2) production. In rats ventilated with VT 20 ml/kg, there was pulmonary edema formation that was significantly increased by hyperoxia. Total lung neutrophil infiltration and MIP-2 in bronchoalveolar lavage (BAL) fluid were significantly elevated, in animals exposed to high VT both on room air (RA) and with hyperoxia. Hyperoxia markedly augmented the migration of neutrophils into the alveoli. Anti-MIP-2 antibody blocked migration of neutrophils into the alveoli in RA by 51% and with hyperoxia by 65%. We concluded that neutrophil migration into the alveoli was dependent on stretch-induced MIP-2 production. Hyperoxia significantly increased edema formation and neutrophil migration into the alveoli with VT 20 ml/kg, although BAL MIP-2 levels were nearly identical to VT 20 ml/kg with RA, suggesting that other mechanisms may be involved in hyperoxia-augmented neutrophil alveolar content in VILI.     

糖皮质激素对肺泡巨噬细胞趋化活性的影响

采用测定肺泡巨噬细胞(AM)趋化性和AM产生中性粒细胞趋化因子的实验技术。观察到糖皮质激素体外直接作用于大鼠AM悬液或大鼠皮下注射7d都可抑制AM的趋化性,并且肺灌洗液中的AM数量明显减少;但注射3d组无上述效应。以糖皮质激素处理AM 16h可使AM产生的中性粒细胞趋化因子强度明显降低。结果表明糖皮质激素可抑制AM的趋化性和抑制AM产生中性粒细胞趋化因子。提示这可能是较长期应用糖皮质激素抑制免疫和炎症反应、降低肺部防御能力的部分机理。     

Alveolar macrophage activation is a key initiation signal for acute lung ischemia-reperfusion injury

Lung ischemia-reperfusion (I/R) injury is a biphasic inflammatory process. Previous studies indicate that the later phase is neutrophil-dependent and that alveolar macrophages (AMs) likely contribute to the acute phase of lung I/R injury. However, the mechanism is unclear. AMs become activated and produce various cytokines and chemokines in many inflammatory responses, including transplantation. We hypothesize that AMs respond to I/R by producing key cytokines and chemokines and that depletion of AMs would reduce cytokine/chemokine expression and lung injury after I/R. To test this, using a buffer-perfused, isolated mouse lung model, we studied the impact of AM depletion by liposome-clodronate on I/R-induced lung dysfunction/injury and expression of cytokines/chemokines. I/R caused a significant increase in pulmonary artery pressure, wet-to-dry weight ratio, vascular permeability, tumor necrosis factor (TNF)-alpha, monocyte chemoattractant protein (MCP)-1, and macrophage inflammatory protein (MIP)-2 expression, as well as decreased pulmonary compliance, when compared with sham lungs. After AM depletion, the changes in each of these parameters between I/R and sham groups were significantly attenuated. Thus AM depletion protects the lungs from I/R-induced dysfunction and injury and significantly reduces cytokine/chemokine production. Protein expression of TNF-alpha and MCP-1 are positively correlated to I/R-induced lung injury, and AMs are a major producer/initiator of TNF-alpha, MCP-1, and MIP-2. We conclude that AMs are an essential player in the initiation of acute lung I/R injury.     

Transient neutrophil infiltration after allergen challenge is dependent on specific antibodies and Fc gamma III receptors

Following allergen challenge of sensitized mice, neutrophils are the first inflammatory cells found in bronchoalveolar lavage (BAL) fluid. To determine the underlying mechanism for their accumulation, mice were sensitized to OVA on days 0 and 14, and received, on day 28, a single intranasal challenge (s.i.n.) with either OVA or ragweed. Eight hours after the s.i.n., BAL fluid was obtained. BALB/c mice sensitized and challenged with OVA showed significantly higher total cell counts and numbers of neutrophils in BAL fluid compared to the OVA-sensitized and ragweed-challenged or nonsensitized mice. Levels of neutrophil chemokines in BAL fluid supernatants were markedly elevated in the sensitized and OVA-challenged mice; Fc epsilon RI-deficient mice showed comparable numbers of neutrophils and neutrophil chemokines in BAL fluid after s.i.n. But in sensitized mice lacking the Fc common gamma-chain and B cell-deficient mice, the number of neutrophils and levels of neutrophil chemokines in BAL fluid were significantly lower. Further, mice lacking the FcgammaRIII did not develop this early neutrophil influx. Neutrophil infiltration could be induced in naive mice following intranasal instillation of allergen combined with allergen-specific IgG1. In addition, macrophages from sensitized mice were stimulated with allergen and activated to produce neutrophil chemokines. These results demonstrate that neutrophil influx after allergen challenge requires prior sensitization, is allergen-specific, is mediated through FcgammaRIII, and is dependent on the presence of Ab.     

Analysis of a Panel of 48 Cytokines in BAL Fluids Specifically Identifies IL-8 Levels as the Only Cytokine that Distinguishes Controlled Asthma from Uncontrolled Asthma,and Correlates Inversely with FEV1

We sought to identify cells and cytokines in bronchoalveolar lavage (BAL) fluids that distinguish asthma from healthy control subjects and those that distinguish controlled asthma from uncontrolled asthma. Following informed consent, 36 human subjects were recruited for this study. These included 11 healthy control subjects, 15 subjects with controlled asthma with FEV₁≥80% predicted and 10 subjects with uncontrolled asthma with FEV₁ <80% predicted. BAL fluid was obtained from all subjects. The numbers of different cell types and the levels of 48 cytokines were measured in these fluids. Compared to healthy control subjects, patients with asthma had significantly more percentages of eosinophils and neutrophils, IL-1RA, IL-1α, IL-1β, IL-2Rα, IL-5, IL-6, IL-7, IL-8, G-CSF, GROα (CXCL1), MIP-1β (CCL4), MIG (CXCL9), RANTES (CCL5) and TRAIL in their BAL fluids. The only inflammatory markers that distinguished controlled asthma from uncontrolled asthma were neutrophil percentage and IL-8 levels, and both were inversely correlated with FEV₁. We examined whether grouping asthma subjects on the basis of BAL eosinophil % or neutrophil % could identify specific cytokine profiles. The only differences between neutrophil-normal asthma (neutrophil≤2.4%) and neutrophil-high asthma (neutrophils%>2.4%) were a higher BAL fluid IL-8 levels, and a lower FEV₁ in the latter group. By contrast, compared to eosinophil-normal asthma (eosinophils≤0.3%), eosinophil-high asthma (eosinophils>0.3%) had higher levels of IL-5, IL-13, IL-16, and PDGF-bb, but same neutrophil percentage, IL-8, and FEV₁. Our results identify neutrophils and IL-8 are the only inflammatory components in BAL fluids that distinguish controlled asthma from uncontrolled asthma, and both correlate inversely with FEV₁.     

Mouse neutrophils require JNK2 MAPK for Toxoplasma gondii-induced IL-12p40 and CCL2/MCP-1 release

The MAPK family member JNK/stress-activated MAPK (SAPK) is involved in extracellular stress and proinflammatory cytokine responses, including production of cytokines such as IL-12. The JNK1 and 2 isoforms are widely expressed, but JNK3 is largely restricted to tissues of the brain, testis, and heart. In this study, we focus on mouse neutrophils, a cell type in which JNK/SAPK expression and activity has been given little study. We used Western blot analysis to examine expression patterns of JNK/SAPK in wild-type and JNK2-/- polymorphonuclear leukocytes (PMN). Surprisingly, neutrophils displayed a major deficiency in JNK1 expression, in contrast to macrophages that expressed high levels of both JNK1 and JNK2 MAPK. JNK1 expression was steadily reduced during the neutrophil maturation in bone marrow. We used PMN infection with the protozoan parasite Toxoplasma gondii to determine whether neutrophil JNK2 was functional. The parasite induced rapid JNK2 phosphorylation and intracellular FACS staining demonstrated preferential activation in infected neutrophils. Use of JNK2-/- neutrophils revealed that this MAPK family member was required for PMN IL-12p40 and CCL2/MCP-1 production. The chemotactic response displayed a minor JNK2 dependence but phagocytosis and oxidative burst activity did not require this MAPK. These findings are important because they demonstrate 1) a previously unrecognized unusual JNK expression pattern in mouse neutrophils, 2) JNK2 in PMN is activated by Toxoplasma invasion, and 3) a requirement for JNK2 in PMN IL-12p40 and CCL2/MCP-1 production in response to a microbial pathogen.     

The expression of IL-20 and IL-24 and their shared receptors are increased in rheumatoid arthritis and spondyloarthropathy

The purpose of this study was to analyze the expression of the two proinflammatory cytokines IL-20 and IL-24 and their shared receptors in rheumatoid arthritis and spondyloarthropathy. IL-20 was increased in plasma of rheumatoid arthritis patients compared with osteoarthritis patients and IL-24 was increased in synovial fluid and plasma of rheumatoid arthritis and spondyloarthropathy patients compared with osteoarthritis patients. IL-20 and IL-24 mRNA was only present at low levels in the synovium. In the synovial membrane, IL-20 protein was present in mononuclear cells and neutrophil granulocytes whereas IL-24 protein was observed in endothelial cells and mononuclear cells. IL-20 receptor type 1 and IL-22 receptor were expressed by granulocytes in the synovial fluid. In synovial fluid mononuclear cell cultures, stimulation with recombinant human IL-20 or recombinant human IL-24 induced monocyte chemoattractant protein 1 (CCL2/MCP-1) secretion, but not tumour necrosis factor alpha mRNA synthesis or IL-6 secretion. Both IL-20 and IL-24 showed correlations to CCL2/MCP-1 in plasma from rheumatoid arthritis and spondyloarthropathy patients. This study associates IL-20 and IL-24 to the synovium of rheumatoid arthritis and spondyloarthropathy and results indicate that the two cytokines contribute to disease pathogenesis through recruitment of neutrophil granulocytes and induction of CCL2/MCP-1.     

Insulin regulation of MCP-1 in human adipose tissue of obese and lean women

CCL2 (MCP-1, monocyte chemoattractant protein 1) and CCL3 (MIP-1alpha, macrophage inflammatory protein 1alpha) are required for macrophage infiltration in adipose tissue. Insulin increases CCL2 expression in adipose tissue and in serum more in insulin-resistant obese than in insulin-sensitive lean mice, but whether this is true in humans is unknown. We compared basal expression and insulin regulation of CCL2 and CCL3 in adipose tissue and MCP-1 and MIP-1alpha in serum between insulin-resistant and insulin-sensitive human subjects. Subcutaneous adipose tissue biopsies and blood samples were obtained before and at the end of 6 h of in vivo euglycemic hyperinsulinemia (maintained by the insulin clamp technique) in 11 lean insulin-sensitive and 10 obese insulin-resistant women, and before and after a 6-h saline infusion in 8 women. Adipose tissue mRNA concentrations of monocyte/macrophage markers CD68, EMR1, ITGAM, ADAM8, chemokines CCL2 and CCL3, and housekeeping gene ribosomal protein large P0 (RPLP0) were measured by means of real-time PCR at baseline. In addition, mRNA concentrations of CCL2, CCL3, and RPLP0 were measured after insulin infusion. Levels of MCP-1 and MIP-1alpha were determined in serum, and protein concentration of MCP-1 was determined in adipose tissue at baseline and after insulin infusion. Basally, expression of the macrophage markers CD68 and EMR1 were increased in adipose tissue of insulin-resistant subjects. Insulin increased MCP-1 gene and protein expression significantly more in the insulin-resistant than in the insulin-sensitive subjects. Basally expression of CCL2 and CCL3 and expression of macrophage markers CD68 and ITGAM were significantly correlated. In serum, MCP-1 decreased significantly in insulin-sensitive but not insulin-resistant subjects. MIP-1alpha was undetectable in serum. Insulin regulation of CCL2 differs between insulin-sensitive and -resistant subjects in a direction that could exacerbate adipose tissue inflammation.     

IFN-gamma-mediated survival enables human neutrophils to produce MCP-1/CCL2 in response to activation by TLR ligands

TLRs are key elements of the pathogen recognition mechanism used by the host immune system. Neutrophils express almost all TLRs, and activation of TLRs, such as TLR2 and TLR4, has been shown to induce the production of proinflammatory cytokines and chemokines, potentially linking innate and adaptive immunity. In the present study, we investigated whether activation of TLRs induces neutrophil production of MCP-1/CCL2, a key mediator involved in the development of adaptive immunity. Activation of neutrophils with LPS, lipoteichoic acid, or N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-Cys-[S]-Ser-[S]-Lys did not induce significant MCP-1 production and release; however, the Th1 cytokine IFN-gamma dramatically up-regulated MCP-1 production in cells activated with each TLR ligand. The majority of MCP-1 was released between 24 and 48 h of culture, indicating that this is a late event. The effect of IFN-gamma appeared to be due to its antiapoptotic effect, but not priming effect, revealing a biological consequence of IFN-gamma-induced neutrophil survival. Although IFN-gamma failed to protect neutrophils from cell death at a higher dose of LPS, the p38 MAPK inhibitor SB203580 dramatically increased MCP-1 release and neutrophil survival at this LPS concentration. Thus, p38 MAPK plays a previously uncharacterized role in neutrophil function. Taken together, our results indicate that human neutrophils produce MCP-1 in a Th1 microenvironment and this neutrophil-derived MCP-1 potentially amplifies the development of Th1 adaptive responses.     

Antimacrophage chemokine treatment prevents neutrophil and macrophage influx in hyperoxia-exposed newborn rat lung

Macrophage-derived cytokines may provoke the inflammatory response in lung injury. Because macrophage influx is a prominent feature of the cellular inflammatory response accompanying the development of bronchopulmonary dysplasia, we hypothesized that blocking macrophage influx would reduce overall cellular influx and oxidative damage. Newborn rats were exposed at birth to 95% O(2) or air for 1 wk, and hyperoxia-exposed pups were injected with anti-monocyte chemoattractant protein-1 (MCP-1) or IgG control on days 3-5. MCP-1 was increased in bronchoalveolar lavage fluid and in histological sections from the 95% O(2)-exposed, IgG-injected pups compared with air-exposed controls. At 1 wk, anti-MCP-1-treated pups had reduced leukocyte numbers, both macrophages and neutrophils, in bronchoalveolar lavage fluid compared with IgG-treated controls. Cytokine-induced neutrophil chemoattractant-1, the rat analog of IL-8, was not significantly decreased in lavage fluid but was reduced in lung cells in anti-MCP-1-treated pups. Tissue carbonyls, a measure of protein oxidation, were decreased in anti-MCP-1-treated pups. Anti-MCP-1 treatment prevented neutrophil influx and reduced protein oxidation in hyperoxia-exposed newborn rats.     

Monocyte chemoattractant protein-1/CC chemokine ligand 2 enhances apoptotic cell removal by macrophages through Rac1 activation

Apoptotic cell removal (efferocytosis) is an essential process in the regulation of inflammation and tissue repair. We have shown that monocyte chemoattractant protein-1/CC chemokine ligand 2 (MCP-1/CCL2) enhances efferocytosis by alveolar macrophages in murine bacterial pneumonia. However, the mechanism by which MCP-1 exerts this effect remains to be determined. Here we explored that hypothesis that MCP-1 enhances efferocytosis through a Rac1/phosphatidylinositol 3-kinase (PI3-kinase)-dependent mechanism.We assessed phagocytosis of apoptotic cells by MCP-1 treated murine macrophages in vitro and in vivo. Rac activity in macrophages was measured using a Rac pull down assay and an ELISA based assay (GLISA). The downstream Rac1 activation pathway was studied using a specific Rac1 inhibitor and PI3-kinase inhibitor in in vitro assays.MCP-1 enhanced efferocytosis of apoptotic cells by murine alveolar macrophages (AMs), peritoneal macrophages (PMs), the J774 macrophage cell line (J774s) in vitro, and murine AMs in vivo. Rac1 activation was demonstrated in these cell lines. The effect of MCP-1 on efferocytosis was completely negated by the Rac1 inhibitor and PI3-kinase inhibitor.We demonstrated that MCP-1 enhances efferocytosis in a Rac1-PI3 kinase-dependent manner. Therefore, MCP-1-Rac1-PI3K interaction plays a critical role in resolution of acute lung inflammation.     

Study of bronchoalveolar lavage fluid in paracoccidioidomycosis: cytopathology and alveolar macrophage function in response to gamma interferon; comparison with blood monocytes

Patients with paracoccidioidomycosis (PCM) present marked involvement of the lungs during the course of the mycosis. The purpose of this work was to obtain bronchoalveolar lavage (BAL) fluid from these patients to study the cytopathology, TNF levels and the oxidative and fungicidal response of alveolar macrophages (AMs) to in vitro incubation with recombinant IFN-gamma. To compare the lung and blood compartments, these determinations were also made in plasma and blood monocytes (BMs) obtained from the same patients. The cytopathology of BAL fluid revealed a predominance of macrophages, but with the presence of neutrophil exudation, and rare lymphocytes and epithelioid and giant cells. Comparison of the oxidative status and fungicidal activity of AMs and circulating BMs demonstrated that both cell types are highly activated for these two functions when compared to control cells. However, TNF levels were higher in BAL fluid than in plasma. The possible mechanisms involved in the hyperresponsiveness of cells from PCM patients are discussed.     

Macrophage derived chemokine (CCL22), thymus and activation-regulated chemokine (CCL17), and CCR4 in idiopathic pulmonary fibrosis

Background

Idiopathic pulmonary fibrosis (IPF) is a chronically progressive interstitial lung disease of unknown etiology. Previously, we have demonstrated the selective upregulation of the macrophage-derived chemokine CCL22 and the thymus activation-regulated chemokine CCL17 among chemokines, in a rat model of radiation pneumonitis/pulmonary fibrosis and preliminarily observed an increase in bronchoalveolar (BAL) fluid CCL22 levels of IPF patients.

Methods

We examined the expression of CCR4, a specific receptor for CCL22 and CCL17, in bronchoalveolar lavage (BAL) fluid cells, as well as the levels of CCL22 and CCL17, to elucidate their pathophysiological roles in pulmonary fibrosis. We also studied their immunohistochemical localization.

Results

BAL fluid CCL22 and CCL17 levels were significantly higher in patients with IPF than those with collagen vascular diseases and healthy volunteers, and there was a significant correlation between the levels of CCL22 and CCL17 in patients with IPF. CCL22 levels in the BAL fluid did not correlate with the total cell numbers, alveolar lymphocytes, or macrophages in BAL fluid. However, the CCL22 levels significantly correlated with the numbers of CCR4-expressing alveolar macrophages. By immunohistochemical and immunofluorescence analysis, localization of CCL22 and CCR4 to CD68-positive alveolar macrophages as well as that of CCL17 to hyperplastic epithelial cells were shown. Clinically, CCL22 BAL fluid levels inversely correlated with DLco/VA values in IPF patients.

Conclusion

We speculated that locally overexpressed CCL22 may induce lung dysfunction through recruitment and activation of CCR4-positive alveolar macrophages.     

Evaluation of inflammatory cytokine secretion by human alveolar macrophages

The alveolar macrophage (AM) secretes interleukin 1beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-8 (IL-8), all of them inflammatory cytokines involved in the pathogenesis of many lung diseases. The aim of the present work was to evaluate the basal and stimulated secretion of these cytokines by human AMs. Human AMs were collected by bronchoalveolar lavage (BAL) from four healthy controls and 13 patients with diffuse interstitial lung disease (five cases of sarcoidosis, three of hypersensitivity pneumonitis and five of idiopathic pulmonary fibrosis). AMs were cultured in the presence or absence of different concentrations of lipopolysaccharide (LPS), phorbolmyristate and gamma-interferon. IL-1beta, TNF-alpha, IL-6 and IL-8 levels were measured in BAL fluid and culture supernatant using specific enzyme-linked immunosorbent assays. The substance found to stimulate the secretion of inflammatory cytokines to the greatest extent was LPS at a concentration of 10 microg/ml. Regarding the secretion of IL-1beta, four observations were of interest: basal secretion was very low; LPS exerted a potent stimulatory effect; considerable within-group variability was observed; and there were no significant differences in the comparisons among groups. With respect to TNF-alpha secretion, the results were similar. The only striking finding was the higher basal secretion of this cytokine with respect to that of IL-1beta. Regarding the secretion of IL-6, the same pattern followed by TNF-alpha was found. However, it should be stressed that the increase induced by LPS was smaller than in the two previous cytokines. Regarding the secretion of IL-8, three findings were patent: the strong basal secretion of this cytokine; the moderate increase induced by LPS; and the existence of significant differences among the different groups with respect to the stimulated secretion of this cytokine, which reached maximum values in patients with idiopathic pulmonary fibrosis. Finally, it should be noted that the pattern of cytokines observed in the BAL fluid was similar to that found in cultured AM supernatants. The pattern of inflammatory cytokine secretion by AMs differs from that of other cells of the mononuclear phagocyte system (MPS). In this sense. AMs secrete low amounts of IL-1, moderate amounts of TNF-alpha and IL-6, and high quantities of IL-8. Adherence is an important stimulus in the secretion of these molecules and LPS elicits an increased secretion inverse to the basal secretion. There is considerable individual variability in the secretion of inflammatory cytokines by the AMs of patients with interstitial lung disease and the AMs of these patients are primed in vivo for the secretion of these cytokines. The results of our study, carried out in vitro, can be extrapolated to the in vivo setting.