The role of Toll-like receptors (TLRs) in innate immunity to Legionella pneumophila, a gram-negative facultative intracellular bacterium, was studied by using bone marrow-derived macrophages and dendritic cells from TLR2-deficient (TLR2 ؊/؊ ), TLR4 ؊/؊ , and wild-type (WT) littermate (C57BL/6 ؋ 129Sv) mice. Intracellular growth of L. pneumophila was enhanced within TLR2 ؊/؊ macrophages compared to WT and TLR4؊/؊ macrophages. There was no difference in the bacterial growth within dendritic cells from WT and TLR-deficient mice. Production of interleukin-12p40 (IL-12p40) and IL-10 after infection with L. pneumophila was attenuated in TLR2 ؊/؊ macrophages compared to WT and TLR4 ؊/؊ macrophages. Induction of IL-12p40, IL-10, and tumor necrosis factor alpha secretion from macrophages by the L. pneumophila dotO mutant, which cannot multiply within macrophages, and heat-killed bacteria, was similar to that caused by a viable virulent strain. There was no difference between the WT and its mutants in susceptibility to the cytopathic effect of bacteria. An L. pneumophila sonicated lysate induced IL-12p40 production by macrophages, but that of TLR2 ؊/؊ macrophages was significantly lower than those of WT and TLR4 ؊/؊ macrophages. Treatment of L. pneumophila sonicated lysate with proteinase K and heating did not abolish TLR2-dependent IL-12p40 production. Our results show that TLR2, but not TLR4, is involved in murine innate immunity against L. pneumophila, although other TLRs may also contribute to innate immunity against this organism.Legionella pneumophila is the major etiologic agent of Legionnaires' disease, a potentially fatal type of pneumonia affecting immunocompromised and immunocompetent subjects (28,30). This gram-negative bacterium can multiply within the mononuclear cells in vivo and in vitro (6, 14) and evades phagosome-lysosome fusion within these cells (3). Several L. pneumophila virulence factors that facilitate intracellular growth have been identified in screenings with macrophages or in an in vivo screening system with signature-tagged mutagenesis (12). One important set of virulence factors is the dot/icm system, which is a type IV secretion system and is required for evasion of phagosome-lysosome fusion (5, 37, 38) and for the establishment of phagosomes permissive for the growth of L. pneumophila within them (8). However, the effector molecule(s) of this system remains undetermined. The innate immunity to L. pneumophila has been extensively studied. The A/J mouse strain is permissive for the intracellular growth of L. pneumophila (48), whereas other inbred strains of mice, such as BALB/c (49), 57BL/6, and 129X1 (10), are not. In terms of permissiveness of A/J macrophages, the genetic determinant of this permissiveness has been confirmed to be within the Lgn locus, and specifically the Bircle/Naip5 gene (4, 9, 10, 40, 52, 53). However, the role of this gene in regulation of intracellular growth of the bacterium is still unknown.Toll-like receptors (TLRs) have been identified as receptors of pathogen-as...

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We developed a new simple assay for the quantitation of the activities of drugs against intracellular Legionella pneumophila. The cells of a murine macrophage-like cell line (J774.1 cells) allowed the intracellular growth and replication of the bacteria, which ultimately resulted in cell death. The infected J774.1 cell monolayers in 96-well microplates were first treated with antibiotics and were further cultured for 72 h. The number of viable J774.1 cells in each well was quantified by a colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and an enzyme-linked immunosorbent assay reader. The number of growing bacteria in each well was also determined by counting the numbers of CFU on buffered charcoal yeast extract-α agar plates. Viable J774.1 cell counts, determined by the colorimetric assay, were inversely proportional to the number of intracellular replicating bacteria. The minimum extracellular concentrations (MIECs) of 24 antibiotics causing inhibition of intracellular growth of L. pneumophila were determined by the colorimetric assay system. The MIECs of beta-lactams and aminoglycosides were markedly higher than the MICs in buffered yeast extract-α broth. The MIECs of macrolides, fluoroquinolones, rifampin, and minocycline were similar to the respective MICs. According to their intracellular activities, clarithromycin and sparfloxacin were the most potent among the macrolides or fluoroquinolones tested in this study. Our results indicated that the MTT assay system allows comparative and quantitative evaluations of the intracellular activities of antibiotics and efficient processing of a large number of samples.

BackgroundHepatocyte growth factor (HGF) is known to be involved in the resolution of pulmonary inflammation and repair of acute lung injury. Legionella pneumonia is sometimes complicated by acute lung injury. Our study aimed to determine the role of serum HGF levels in Legionella pneumonia.MethodsSera from patients with Legionella pneumonia (42 cases), other bacterial pneumonia (33 cases), pulmonary tuberculosis (19 cases), and normal controls (29 cases) were collected. The serum HGF levels for each serum sample were determined by sandwich ELISA. Clinical and laboratory data were collected by reviewing the medical charts.ResultsSerum HGF levels were higher in patients with Legionella pneumonia than in those with other bacterial pneumonia, pulmonary tuberculosis, and controls. The HGF levels were compared with white blood cell counts, C-reactive protein, Alanine amino- transferase, and lactate dehydrogenase (LDH). The HGF levels were correlated to serum LDH levels. Moreover, serum HGF levels were significantly higher in non-survivors than in survivors.ConclusionsHGF levels increased in severer pneumonia caused by Legionella, suggesting that HGF might play a significant role in the Legionella pneumonia.

A 48-year-old womanwas admitted to our hospital with high fever, chills, cough, and exertional dyspnea. Onadmission, the chest roentgenogram and computed tomography scan showed bilateral alveolar infiltration in the middle and lower lung fields. Microscopic examination of the bronchial lavage fluid showed flower cells typical for adult T-cell leukemia (ATL) and cysts of Pneumocystis carinii, and Legionella pneumophila serogroup 1 grew on buffered charcoal yeast extract (BCYE)-a agar. The patient was successfully treated with antibiotics including trimethoprim/ sulfamethoxazole, erythromycin, and sparfloxacin. Remission of ATLwas achieved after three courses of antileukemic chemotherapy. Mixedinfection of opportunistic pathogens should be considered in patients with ATL.

A 69-year-old man developed a cough and fever during treatment with corticosteroid (P.O and external use) for erythroderma. Chest X-ray films revealed a consolidation shadow in the right upper lung field. Initial treatment with sulbactam sodium/ampicillin followed by imipenem/ cilastatin was not effective. A urinary antigen test for Legionella was positive, making for a diagnosis of Legionella pneumonia. Intravenous treatment with ciprofloxacin (CPFX) was remarkably effective. His symptoms, chest Xray and laboratory data rapidly improved after its initiation. Our findings strongly suggest that intravenous treatment with fluoroquinolones including CPFXshould also be a first choice for Legionella pneumoniain Japan.

We have evaluated urine specimens of presumptive cases of legionnaires' disease (110 cases, 173 sample), collected in the past eight years (April, 1990-August, 1998) with the Binax EIA kit which detects the soluble antigen of Legionella pneumophila serogroup (SG) 1, and the Biotest EIA kit which detects Legionella species. Seven cases (19 specimens) were positive for the Binax EIA kit, and nine cases (22 specimens) were positive for the Biotest EIA kit. The sensitivity for culture, PCR, IFA method were 100%, 100%, and 50%, the specificity for these method were 93%, 97.1%, and 90% respectively. Overall agreements for these method were 93.5%, 97.4%, 86.8%, these results suggested that the urinary antigen detection test had high sensitivity and specificity. Our study indicated that concentrated urine samples increase sensitivity. We also evaluated the capabilities of both EIAs to detect soluble antigens were extracted from bacterial suspension of 18 strains of 5 Legionella species by heating. Both assays detected L. pneumophila serogroups 1 to 14, L. bozemanii. The Binax EIA proved to be useful as the Biotest EIA for diagnosis of legionellosis caused by Legionella species and serogroups other than L. pneumophila serogroup 1. Some cases have been shown to excrete antigen for prolonged period of times despite recovery from infection, so that the patient's history should be sought. The urine antigen detection EIA methods proved to be rapid and easy to use, detect antigen in the early stage of the disease with high sensitivity and specificity. Its use for the definition of legionellosis should be considered in Japan.