Bronchoalveolar Lavage Mononuclear Cells Research Articles

Enhanced antigen presenting and T cell functions during late-phase allergic responses in the lung.

Allergic inflammation is a common feature of asthma and may contribute to both development and perpetuation of disease. The interaction of antigen-presenting cells (APC) with sensitized helper T lymphocytes (TC) producing Th2 cytokines may determine the inflammatory response. Recruitment of APC and TC to the lung during allergic responses has been demonstrated, but functional studies in humans have been limited. This study examined the function of APC and TC accumulating at sites of inflammation after segmental allergen challenge (SAC). Fifteen allergic patients underwent SAC, and cells from bronchoalveolar lavage (BAL) were collected after 24 hours. APC and TC from the blood and BAL were purified based on expression of the monocyte marker, CD14; the plasmacytoid dendritic cell (pDC) marker, BDCA4, identifying neuropilin-1 (NRP1); and the helper T cell marker, CD4. Functional activity was assessed using allergen-induced T cell proliferation. Flow cytometry identified cells expressing CD14 and NRP1. SAC resulted in a 12-fold increase in mononuclear cells having the morphologic appearance of blood monocytes. Most of these cells co-expressed CD14 and NRP1. After saline challenge, BAL mononuclear cells demonstrated little APC function. Following SAC, BAL mononuclear cells showed function equal to pDC from blood and greater than blood monocytes. Purified NRP1+ cells from BAL had even greater function than pDC cells from blood (P = .008). Using consistent sources of APC, enhanced proliferation of TC from lung compared to blood was also demonstrated (P = .002). The marked increase in APC function for allergen-specific TC proliferation during allergic inflammation is largely due to the recruitment of monocytes and dendritic cells. There is also an enhanced response in the lung TC population, consistent with recruitment of allergen-specific T cells. Interactions between recruited APC and TC may occur as an early event promoting allergic airway inflammation.

Increased frequencies of pulmonary regulatory T-cells in latentMycobacterium tuberculosisinfection

Regulation of specific immune responses following exposure to Mycobacterium tuberculosis in humans and the role of regulatory T (Treg) cells in the immune control of latent infection with M. tuberculosis are incompletely understood. Latent infection was assayed by an interferon-γ release assay (IGRA) in healthcare workers regularly exposed to tuberculosis (TB) patients and in household TB contacts in Germany. Immunophenotypes of bronchoalveolar lavage (BAL) mononuclear cells and peripheral blood mononuclear cells (PBMCs) were analysed by fluorescence-activated cell sorting. All TB contacts with latent infection (n=15) had increased (p<0.0001) frequencies of CD4+ CD25+ CD127- Treg cells (median 2.12%, interquartile range (IQR) 1.63-3.01%) among BAL mononuclear cells compared with contacts (n=25) with negative IGRA results (median 0.68%, IQR 0.32-0.96%) No differences were seen when PBMC immunophenotypes of IGRA+ and IGRA- TB contacts were compared (IGRA+ median 9.6%, IQR 5.9-10.1%; IGRA- median 7.7%, IQR 4.6-11.3%; p=0.47). Five out of 25 contacts with negative blood IGRAs showed a positive IGRA from BAL cells, possibly indicating a limited local immune response. In Germany, latent infection with M. tuberculosis, as defined by a positive M. tuberculosis-specific IGRA response on cells from the peripheral blood, is characterised by an increased frequency of Treg cells in the BAL.

Antimycobacterial immune responses in patients with pulmonary sarcoidosis

Sarcoidosis is a multisystem granulomatous disease of unknown origin. Pathogenetic involvement of Mycobacterium tuberculosis has frequently been discussed in the aetiology of sarcoidosis; however, studies still remain contradictory. We addressed the question of mycobacterial involvement in the pathogenesis of sarcoidosis by analysing cellular immune responses to mycobacterial antigens. We examined the interferon (IFN)-gamma production by enzyme-linked immunospot in response to purified protein derivate (PPD) mycobacterial-specific antigen early secretory antigenic target (ESAT)-6 and culture filtrate protein (CFP)-10 by peripheral blood mononuclear cells (PBMCs) and bronchoalveolar-lavage mononuclear cells (BALMCs) of patients with pulmonary sarcoidosis, smear-negative tuberculosis and controls. Release of IFN-gamma in response to ex vivo contact with PPD, ESAT-6 or CFP-10 by BALMC and PBMC were comparable among patients with sarcoidosis and controls (PBMC P = 0.2326; BALMC P = 0.1767) and were less frequently observed in both groups compared to patients with tuberculosis (BALMC P < 0.05; PBMC P < 0.0001). Within PBMC, the immunophenotype of sarcoidosis patients differed from that of patients with tuberculosis, as well as from that of controls, while within BALMC it resembled that of patients with tuberculosis. In contrast to patients with tuberculosis, the frequency of mycobacteria-specific local and systemic immune responses is not elevated in patients with sarcoidosis when compared to controls. The immunophenotype represents the local resemblance of the granulomatous reaction underlying tuberculosis and sarcoidosis while showing systemical difference. These observations do not support a role of an infection with M. tuberculosis in the pathogenesis of sarcoidosis.

Bronchoalveolar Lavage Enzyme-linked Immunospot for a Rapid Diagnosis of Tuberculosis

The rapid diagnosis of pulmonary tuberculosis (TB) is difficult when acid fast bacilli (AFB) cannot be detected in sputum smears. Following a proof of principle study, we examined in routine clinical practice whether individuals with sputum AFB smear-negative TB can be discriminated from those with latent TB infection by local immunodiagnosis with a Mycobacterium tuberculosis-specific enzyme-linked immunospot (ELISpot) assay. Subjects suspected of having active TB who were unable to produce sputum or with AFB-negative sputum smears were prospectively enrolled at Tuberculosis Network European Trialsgroup centers in Europe. ELISpot with early-secretory-antigenic-target-6 and culture-filtrate-protein-10 peptides was performed on peripheral blood mononuclear cells (PBMCs) and bronchoalveolar lavage mononuclear cells (BALMCs). M. tuberculosis-specific nucleic acid amplification (NAAT) was performed on bronchoalveolar lavage fluid. Seventy-one of 347 (20.4%) patients had active TB. Out of 276 patients who had an alternative diagnosis, 127 (46.0%) were considered to be latently infected with M. tuberculosis by a positive PBMC ELISpot result. The sensitivity and specificity of BALMC ELISpot for the diagnosis of active pulmonary TB were 91 and 80%, respectively. The BALMC ELISpot (diagnostic odds ratio [OR], 40.4) was superior to PBMC ELISpot (OR, 10.0), tuberculin skin test (OR, 7.8), and M. tuberculosis specific NAAT (OR, 12.4) to diagnose sputum AFB smear-negative TB. In contrast to PBMC ELISpot and tuberculin skin test, the BALMC ELISpot was not influenced by previous history of TB. Bronchoalveolar lavage ELISpot is an important advancement to rapidly distinguish sputum AFB smear-negative TB from latent TB infection in routine clinical practice.

Antimycobacterial immuneresponses in patients with pulmonary sarcoidosis

Sarcoidosis is a multisystem granulomatous disease of unknown origin. Pathogenetic involvement of Mycobacterium tuberculosis has frequently been discussed, however, studies still remain contradictory. We examined the interferon (IFN)-γ production by enzyme-linked-immunospot in response to purified protein derivate (PPD) and mycobacterial specific antigens early-secretory-target (ESAT)-6 and culture-filtrate-protein (CFP)-10 by peripheral blood mononuclear cells (PBMC) and bronchoalveolar-lavage mononuclear cells (BALMC) of patients with pulmonary sarcoidosis, smear-negative tuberculosis and controls. Release of IFN-γ in response to ex vivo contact with PPD, ESAT-6 or CFP-10 by BALMC and PBMC were comparable among patients with sarcoidosis and controls (PBMC p=0.1630; BALMC p=0.2199) and were less freuqently observed in both groups compared to patients with tuberculosis (BALMC p&lt;0,0131; PBMC p&lt;0.0001). Within PBMC the immunephenotype of sarcoidosis-patients differed from patients with tuberculosis, as well as from controls, while within BALMC it resembled the one of patients with tuberculosis. In contrast to patients with tuberculosis, the frequency of mycobacteria-specific local and systemic immunresponses is not elevated in patients with sarcoidosis when compared to controls. The immunephenotype represents the local resemblance of the granulomatous reaction underlying tuberculosis and sarcoidosis, while showing systemical difference. These observations do not support a role of a mycobacterial infection in the pathogenesis of sarcoidosis.

Safety, Antigenicity, and Efficacy of a Recombinant Coccidioidomycosis Vaccine in Cynomolgus Macaques (Macaca fascicularis)

The safety, immunogenicity and efficacy of recombinant Ag2/PRA106 + CSA chimeric fusion protein (CFP) vaccine in ISS/Montanide adjuvant-administered intramuscular (IM) was assessed in adult female cynomolgus macaques challenged with Coccidioides posadasii. Animals received three immunizations with either 5 microg CFP, 50-microg CFP, or adjuvant alone and were challenged 4 weeks following the final immunization. Although significant antibody response was produced in response to vaccination, there were no discernable adverse effects, suggesting that the vaccine was well tolerated. Upon intratracheal challenge, all animals showed evidence of disease. Two animals that received 5-microg doses of CFP were euthanatized prior to the study's end because of severe symptoms. Animals vaccinated with 50-microg doses of CFP showed evidence of enhanced sensitization compared to adjuvant controls and animals vaccinated with 5-microg doses of CFP. This was based on higher serum anti-CFP titers, enhanced secretion of interferon-gamma (IFN-gamma) from stimulated bronchoalveolar lavage mononuclear cells (BALMC), reduced pulmonary radiologic findings following intratracheal challenge, reduced terminal complement fixation titers, and reduced necropsy findings. Overall the vaccine was well tolerated, induced sensitization, and resulted in a protective response when given at the higher 50-microg dose. Additional experiments may be needed to optimize the vaccination and to confer greater protection against lethal challenge.

RANTES, IFN-γ, CCR1, and CCR5 mRNA Expression in Peripheral Blood, Lymph Node, and Bronchoalveolar Lavage Mononuclear Cells during Primary Simian Immunodeficiency Virus Infection of Macaques

Primary infection of macaques with pathogenic isolates of simian immunodeficiency virus (SIV) (as a model of HIV infection in humans) represents a unique opportunity to study early lentivirus/host interactions. We sought to determine whether there is a temporal relationship linking SIV replication and dissemination and the expression of the chemokine RANTES (regulated upon activation normal T cell expressed and secreted) and the SIV/HIV coreceptor CCR5 in different tissues during acute SIV infection of macaques. Four cynomolgus macaques were inoculated intravenously with a pathogenic primary isolate of SIVmac251. RT-PCR was used to monitor the expression of RANTES and CCR5 mRNA in fresh isolated mononuclear cells from blood, lymph node, and bronchoalveolar lavages. These expressions were compared to those of IFN-γ as an indicator of the development of the immune response and to another receptor for RANTES, CCR1, which is not described as a coreceptor for SIV/HIV-1 entry. An enhancement of CCR1/CCR5 mRNA expression was noticed during primary SIVmac251 infection of macaques, mainly in tissue. In the three different compartments investigated, IFN-γ and RANTES overexpression was noticed by the time of systemic viral replication containment. Our results put CCR5 and RANTES mRNA expression back in the context of inflammatory and immune responses to SIV primary infection.

Transforming growth factor β within fibrotic scleroderma lungs

purpose, patients, and methods: Since transforming growth factor β (TGFβ) has been implicated as an important mediator of pulmonary fibrosis, we measured TGFβ protein and gene expression in alveolar epithelial lining fluid (ELF) of fibrotic scleroderma lungs sampled by bronchoalveolar lavage (BAL). TGFβ protein was qualitatively examined by Western blot analysis, and quantitatively by radioreceptor assays. Gene expression was evaluated in BAL mononuclear cells by Northern blot analysis with quantification of relative gene expression by densitometric analysis of the autoradiograms. results: Normal and scleroderma subjects had a 24-kd protein that comigrated with defined human TGFβ and immunoreacted with anti-TGFβ antibody. The normal population had a significantly higher average TGFβ concentration (705 pM) compared with the scleroderma subjects (177 pM). The TGFβ gene was expressed in amounts that did not significantly differ between the scleroderma and normal groups. On an individual subject basis, the TGFβ concentration variability did not correlate with variations in BAL cellularity or TGFβ gene expression within the recovered mononuclear cells. conclusions: It is concluded that both normal and fibrotic lungs have TGFβ present at the alveolar epithelial surface. However, in the fibrotic scleroderma lungs, TGFβ protein content and gene expression were not increased at the alveolar epithelial surface. The simultaneous analysis of TGFβ protein content, gene expression, and cellular constituents within individual ELF specimens showed that the cellular components of the ELF do not appear to be major determinants of TGFβ protein concentration at the alveolar epithelial surface.

Warm ischemia induces alteration in lung immune cell functions

Warm ischemia is an important factor in early allograft dysfunction. To elucidate cellular events involved in such lung injury, we examined the effects of warm ischemia on the cytotoxic function of lymphocytes retrieved by bronchoalveolar lavage as compared with peripheral blood lymphocytes. Warm ischemia of the lung was induced in eight dogs by crossclamping left hilar structures for 1 hour. Bronchoalveolar cells from ischemia left and unaffected right lungs, as well as blood lymphocytes, were isolated before operation and 2 hours, 72 hours, and 7 days after operation. Lung and blood lymphocytes were assayed for natural killer and lectin-dependent cell-mediated cytotoxicity. Warm ischemia resulted in a significant impairment of natural killer activity within 2 hours of reperfusion (49% of preoperative control cytolysis, p less than 0.01). There was a significant increase in natural killer activity in bronchoalveolar lavage mononuclear cells 72 hours after reperfusion injury (178.4% of preoperative value, p less than 0.01). Interestingly, these functional alterations were not paralleled with changes seen in the peripheral blood lymphocytes or the opposite nonaffected lungs, where the natural killer activity appeared significantly depressed at 72 hours. Similarly, lectin-dependent cell-mediated cytotoxicity was noted to be increased in the bronchoalveolar lavage from the ischemic lung (179.5%, p less than 0.01) but decreased in the bronchoalveolar lavage from the nonaffected lung and peripheral blood lymphocytes at 72 hours after injury. We conclude that warm ischemia is associated with a functional alteration of the local lung immune cells. Such alteration is not observed in cells from the opposite lung or peripheral blood. The observed increase in nonspecific cytotoxicity of bronchoalveolar lymphocytes can be causative in the early damage seen in poorly preserved lung allografts.

Stimulation of bronchoalveolar lavage (BAL) and blood lymphocytes by Kveim antigen, tuberculin and concanavalin A in sarcoidosis

BAL and blood mononuclear cells and their reactivity to Kveim antigen, tuberculin and concanavalin A (Con A) were studied in nine patients with different clinical stages of sarcoidosis. After separation by plastic adherence, non-adherent cells (mainly lymphocytes) were admixed with 10% autologous adherent cells (monocytes/macrophages). After 3 and 6 days' culture with Kveim antigen (1, 10, 100 μg/ml), PPD tuberculin (2.5 μg/ml) and Con A (10, 20, 40 μg/ml) stimulation was measured as incorporation of 14C-thymidine into DNA. Except for occasional reactions the study did not show any unitary significant increase in lymphocyte response to the different concentrations of Kveim antigen in either BAL or blood. For Con A there was a weaker response by BAL-mononuclear cells with no difference between 3 and 6 days, compared with blood where there was an early peak. The lymphocyte reaction to PPD was weak with no difference between blood and BAL.

Immunoregulation in sarcoidosis. Perpetual secretory dysfunction of natural killer-like cells.

Sarcoidosis is an immunopathogenic disorder of uncertain cause. Because the regulation of monocyte mobilization and function may be critical to granuloma formation in this disease, we evaluated production of the cell-directed inhibitor of monocyte leukotaxis (CDI-MLx) by peripheral blood and bronchoalveolar lavage mononuclear cells from these patients. Cells obtained from either source during clinically active disease spontaneously produced this leukotactic regulator in vitro in amounts comparable to those achieved with maximal mitogenic stimulation of normal cells. Plasma leukotactic inhibitory activity and spontaneous inhibitor production were significantly associated. Plasma inhibitory activity and CDI-MLx production were normal in patients whose disease was inactive. Spontaneous production continued for at least 7 days in vitro and could not be attributed to alterations in the absolute numbers of specific mononuclear cell populations. Partitioning of peripheral blood mononuclear cells by a combination of E-rosetting and immunoadherence techniques indicated, however, that CDI-MLx was produced by a subpopulation of natural killer-like cells that formed E-rosettes and bore the OKMI and Leu 7 membrane antigens.