In searching for immunogenic molecules with the potential to induce protective immune responses against tuberculosis, we developed an ex vivo model to study frequency, phenotype, and effector functions of human T lymphocytes recognizing hydrophobic Ags of Mycobacterium tuberculosis (M.Tb). To obtain unbiased results, we characterized T lymphocytes responding to a crude cell wall extract (chloroform methanol extract of M.Tb (M.Tb-CME)) containing a broad spectrum of mycobacterial glycolipids and lipopeptides. A significant proportion of T lymphocytes recognized M.Tb-CME (290 IFN-y(+) T cells/10(5) PBMCs) and developed to effector memory cells as determined by the expression of CD45RO and the chemokine receptors CXCR3 and CCR5. Expanded lymphocytes fulfilled all criteria required for an efficient immune response against tuberculosis: 1) release of macrophage-activating Th1 cytokines and chemokines required for the spatial organization of local immune responses, 2) cytolytic activity against Ag-pulsed macrophages, and 3) recognition of infected macrophages and killing of the intracellular bacteria. Phenotypically, M.Tb-CME-expanded cells were CD4(+) and MHC class II restricted, challenging current concepts that cytotoxic and antimicrobial effector cells are restricted to the CD8(+) T cell subset. Pretreatment of M.Tb-CME with protease or chemical delipidation abrogated the biological activity, suggesting that responses were directed toward mycobacterial lipopeptides. These findings suggest that lipidated peptides are presented by M.Tb-infected macrophages and elicit CD4(+) cytolytic and antimicrobial T lymphocytes. Our data support an emerging concept to include hydrophobic microbial Ags in vaccines against tuberculosis.

Low-dose radiotherapy (LD-RT) is known to exert an anti-inflammatory effect, however, the underlying molecular mechanisms are not fully understood. The manipulation of polymorphonuclear neutrophil (PMN) function and/or recruitment may be one mechanism. Chemokines contribute to this process by creating a chemotactic gradient and by activating integrins. This study aimed to characterize the effect of LD-RT on CCL20 chemokine production and PMN/endothelial cell (EC) adhesion.

Rheumatoid arthritis (RA) is characterized by the recruitment of leukocytes and the accumulation of inflammatory mediators within the synovial compartment. Release of the chemokine CCL18 has been widely attributed to antigen-presenting cells, including macrophages and dendritic cells. This study investigates the production of CCL18 in polymorphonuclear neutrophils (PMN), the predominant cell type recruited into synovial fluid (SF). Microarray analysis, semiquantitative and quantitative reverse transcriptase polymerase chain reaction identified SF PMN from patients with RA as a novel source for CCL18 in diseased joints. Highly upregulated expression of other chemokine genes was observed for CCL3, CXCL8 and CXCL10, whereas CCL21 was downregulated. The chemokine receptor genes were differentially expressed, with upregulation of CXCR4, CCRL2 and CCR5 and downregulation of CXCR1 and CXCR2. In cell culture experiments, expression of CCL18 mRNA in blood PMN was induced by tumor necrosis factor α, whereas synthesis of CCL18 protein required additional stimulation with a combination of IL-10 and vitamin D3. In comparison, recruited SF PMN from patients with RA were sensitized for CCL18 production, because IL-10 alone was sufficient to induce CCL18 release. These results suggest a release of the T cell-attracting CCL18 by PMN when recruited to diseased joints. However, its production is tightly regulated at the levels of mRNA expression and protein synthesis.

http://www.sciencemag.org/cgi/content/full/291/5508/1541 version of this article at: including high-resolution figures, can be found in the online Updated information and services, http://www.sciencemag.org/cgi/content/full/291/5508/1541/DC1 can be found at: Supporting Online Material found at: can be related to this article A list of selected additional articles on the Science Web sites http://www.sciencemag.org/cgi/content/full/291/5508/1541#related-content http://www.sciencemag.org/cgi/content/full/291/5508/1541#otherarticles , 14 of which can be accessed for free: cites 28 articles This article 114 article(s) on the ISI Web of Science. cited by This article has been http://www.sciencemag.org/cgi/content/full/291/5508/1541#otherarticles 48 articles hosted by HighWire Press; see: cited by This article has been http://www.sciencemag.org/cgi/collection/immunology Immunology : subject collections This article appears in the following

Innate immunity provides a first line of host defence against infection through microbial recognition and killing while simultaneously activating a definitive adaptive immune response. Toll-like receptors (TLRs) are principal mediators of rapid microbial recognition and function mainly by detection of structural patterns that do not exist in the host. TLR2 and TLR4 were the first members of this innate immune receptor family to be strongly implicated in antibacterial host defence. Following the initial description of the mammalian TLR family, susceptibility to infection with numerous human microbial pathogens has been intensively studied using mice with engineered deletions of each of these molecules. While it has become quite clear that TLR activation is necessary for optimal host defence, a comprehensive understanding of the mechanisms by which this family of pattern recognition receptors engages protective immunity, particularly the adaptive response, is still evolving.

Einleitung und Methodik: Die Aktivierung von Toll-like Rezeptoren (TLRs) führt zur Abtötung von Mycobacterium tuberculosis (M. Tb) in humanen Alveolarmakrophagen (AM) (Stenger et al., Science, 291, 2001). Ziel der jetzigen Arbeit war die Identifikation der antimikrobiellen Effektormoleküle mittels Microarray-Analysen von AM. Ergebnisse: Die Aktivierung von TLR2 in AM mit Lipopeptiden führte zu einer Hochregulation der mRNA für das Schlüsselenzym der Vitamin D3 Synthese Cyp27B1 (7,4±2,3-fach) und des Vitamin D3 Rezeptors (VDR) (2,3±0,6-fach), der an der Regulation immunmodulatorischer Moleküle beteiligt ist. Agonisten des VDR induzierten die mRNA-Synthese des antimikrobiellen Peptids LL-37 (23±4,9-fach), dessen Wirkungsspektrum auch Mykobakterien einschließt. In AM von 11 Patienten wurde gezeigt, dass Antagonisten des VDR (ZK159222) die TLR-induzierte Expression von LL-37 hemmen. Gleichzeitig wurde dadurch das Wachstum von virulentem M. Tb in AM signifikant gesteigert (Kontrolle: 8,6×106 Bakterien; +10µM Antagonist: 3,1×106 Bakterien). Schlussfolgerung: Unsere Daten beschreiben eine Verbindung zwischen Vitamin D und der erfolgreichen Immunabwehr gegen Mykobakterien und liefern eine Erklärung für die seit langem bekannte Assoziation von VDR-Polymorphismen und dem erhöhten Erkrankungsrisiko für eine Tuberkulose. Denkbar ist, dass durch direkte Zufuhr oder vermehrte Synthese von Vitamin D bei Sonneneinstrahlung ein Heilungseffekt bei Tuberkulosepatienten erzielt werden kann.

ABSTRACT In cystic fibrosis (CF), the condition limiting the prognosis of affected children is the chronic obstructive lung disease accompanied by chronic and persistent infection with mostly mucoid strains of Pseudomonas aeruginosa. The majority of CF patients have antineutrophil cytoplasmic antibodies (ANCA) primarily directed against the bactericidal permeability-increasing protein (BPI) potentially interfering with antimicrobial effects of BPI. We analyzed the expression of BPI in the airways of patients with CF. In their sputum samples or bronchoalveolar lavage specimens, nearly all patients expressed BPI mRNA and protein, which were mainly products of neutrophil granulocytes as revealed by intracellular staining and subsequent flow cytometry. Repeated measurements revealed consistent individual BPI expression levels during several months quantitatively correlating with interleukin-8. In vitro, P. aeruginosa isolates from CF patients initiated the rapid release of BPI occurring independently of protein de novo syntheses. Furthermore, purified natural BPI as well as a 27-mer BPI-derived peptide displayed antimicrobial activity against even patient-derived mucoid P. aeruginosa strains and bacteria resistant against all antibiotics tested. Thus, BPI that is functionally active against mucoid P. aeruginosa strains is expressed in the airways of CF patients but may be hampered by autoantibodies, resulting in chronic infection.

Antimicrobial effector proteins are a key mechanism for the innate immune system to combat pathogens once they infect the host. We report the identification and cloning of the mouse homologue of human bactericidal permeability/increasing protein (BPI). Mouse BPI is constitutively expressed in lymphatic organs and tissues as well as in mouse testis. Upon stimulation with different TLR ligands, mouse BPI is strongly expressed in granulocytes and, surprisingly, in bone marrow-derived dendritic cells. Mouse BPI is most strongly induced by bacterial LPS through a signaling pathway that is completely dependent on TLR4-Toll/IL-1R domain-containing adaptor inducing IFN-beta. Functional studies revealed that mouse BPI does have the potential to neutralize LPS and inhibits bacterial growth. Mouse BPI is expressed in granulocytes and bone marrow-derived dendritic cells, and the transcriptional activation is controlled by TLRs.

ABSTRACT Vaginal infections caused by the opportunistic yeast Candida albicans are a significant problem in women of child-bearing age. Several factors are recognized as playing a crucial role in the pathogenesis of superficial candidiasis; these factors include hyphal formation, phenotypic switching, and the expression of virulence factors, including a 10-member family of secreted aspartic proteinases. In the present investigation, we analyzed the secreted aspartic proteinase gene (SAP) expression profile of C. albicans that is elicited in the course of vaginal infection in mice and how this in vivo expression profile is associated with hyphal formation. We utilized two different genetic reporter systems that allowed us to observe SAP expression on a single-cell basis, a recombination-based in vivo expression technology and green fluorescent protein-expressing Candida reporter strains. Of the six SAP genes that were analyzed (SAP1 to SAP6), only SAP4 and SAP5 were detectably induced during infection in this model. Expression of both of these genes was associated with hyphal growth, although not all hyphal cells detectably expressed SAP4 and SAP5. SAP5 expression was induced soon after infection, whereas SAP4 was expressed at later times and in fewer cells compared with SAP5. These findings point to a link between morphogenetic development and expression of virulence genes during Candida vaginitis in mice, where host signals induce both hyphal formation and expression of SAP4 and SAP5, but temporal gene expression patterns are ultimately controlled by other factors.

Dendritic cells (DCs) are a key part of host defense against microbial pathogens, being part of the innate immune system, but also instructing the adaptive T cell response. This study was designed to evaluate whether human DCs directly contribute to innate immunity by killing intracellular bacteria, using tuberculosis as a model. DCs were detected in bronchoalveolar lavage samples indicating that DCs are available for immediate interaction with Mycobacterium tuberculosis ( M. Tb ) after inhalation of the pathogen. The phenotype of DC in bronchoalveolar lavage closely resembles monocyte-derived immature DC (iDC) according to the expression of CD1a, CD83, and CCR7. The antimicrobial activity of iDC against intracellular M. Tb inversely correlated with TNF- (cid:1) -release and was enhanced by treatment with anti-TNF- (cid:1) Abs. Differentiation of iDC into mature DC by addition of TNF- (cid:1) or activation via Toll-like receptors further reduced killing of M. Tb . The antibacterial activity against intracellular M. Tb of all DCs was significantly lower than alveolar macrophages. Therefore, the maintenance of a pool of DCs at the site of disease activity in tuberculosis, and the maturation of these DC by TNF- (cid:1) provides a mechanism by which M. Tb escapes the innate immune system. The Journal of Immunology, 2005, 174: 4203–4209.

yme disease caused by three genospecies of the spirochete Borrelia burgdorferi sensu lato is the most common tick- borne disease in humans in the Northern Hemisphere. In- fection with B. burgdorferi provokes a multiorgan inflammatory ailment, but the precise underlying mechanisms are not well un- derstood (1). The disease is characterized by some or all of the following clinical manifestations: an initial erythematous rash, lymphocytoma cutis, acrodermatitis chronica atrophicans, neuro- logical disorders, carditis, and, especially, arthritis. Intermittent or chronic oligoarticular arthritis primarily affecting large joints such as the knees is a debilitating late complication. Although most patients with acute Lyme arthritis can be treated effectively with antibiotics, 10% of patients have persistent knee swelling for months to years after 2 mo of medication. This condition has been termed antibiotic treatment-resistant Lyme arthritis (2). In a murine model of Lyme borreliosis, used to delineate mech- anisms of inflammation and protective immunity, genetically sus- ceptible mouse strains like C3H/He develop severe carditis and arthritis when infected with B. burgdorferi. The disease pattern arising in joints of mice resembles human Lyme arthritis, with edema, hyperproliferation of synovial cells, as well as massive infiltration of neutrophils and monocytes, but markedly few lym- phocytes into tissue and joint space (3). The severity of inflam- mation peaks at 2-3 wk after infection and regresses spontane- ously in the presence of B. burgdorferi-specific Abs. Leukocytes are recruited from circulating blood to the inflamed tissue by sequential adhesive interactions with the endothelium in

After infection with Borrelia burgdorferi, humans and mice under certain conditions develop arthritis. Initiation of inflammation is dependent on the migration of innate immune cells to the site of infection, controlled by interactions of a variety of adhesion molecules. In this study, we used the newly synthesized compound S18407, which is a prodrug of the active drug S16197, to analyze the functional importance of α4β1-dependent cell adhesion for the development of arthritis and for the antibacterial immune response. S16197 is shown to interfere specifically with the binding of α4β1 integrin to its ligands VCAM-1 and fibronectin in vitro. Treatment of B. burgdorferi-infected C3H/HeJ mice with the α4β1 antagonist significantly ameliorated the outcome of clinical arthritis and the influx of neutrophilic granulocytes into ankle joints. Furthermore, local mRNA up-regulation of the proinflammatory mediators IL-1, IL-6, and cyclooxygenase-2 was largely abolished. Neither the synthesis of spirochete-specific Igs nor the development of a Th1-dominated immune response was altered by the treatment. Importantly, the drug also did not interfere with Ab-mediated control of spirochete load in the tissues. These findings demonstrate that the pathogenesis, but not the protective immune response, in Lyme arthritis is dependent on the α4β1-mediated influx of inflammatory cells. The onset of inflammation can be successfully targeted by treatment with S18407.

SAP7 of Candida albicans is induced after vaginal infection of mice. Conversely, virulence during vaginal infection was not affected in a Deltasap7/Deltasap7 mutant strain. Only a partial virulence phenotype was detectable after intravenous injection. In conclusion, SAP7 expression does not correlate with C. albicans virulence in mice.

After infection with Borrelia burgdorferi, humans and mice under certain conditions develop arthritis. Initiation of inflammation is dependent on the migration of innate immune cells to the site of infection, controlled by interactions of a variety of adhesion molecules. In this study, we used the newly synthesized compound S18407, which is a prodrug of the active drug S16197, to analyze the functional importance of alpha4beta1-dependent cell adhesion for the development of arthritis and for the antibacterial immune response. S16197 is shown to interfere specifically with the binding of alpha4beta(1 integrin to its ligands VCAM-1 and fibronectin in vitro. Treatment of B. burgdorferi-infected C3H/HeJ mice with the alpha4beta1 antagonist significantly ameliorated the outcome of clinical arthritis and the influx of neutrophilic granulocytes into ankle joints. Furthermore, local mRNA up-regulation of the proinflammatory mediators IL-1, IL-6, and cyclooxygenase-2 was largely abolished. Neither the synthesis of spirochete-specific Igs nor the development of a Th1-dominated immune response was altered by the treatment. Importantly, the drug also did not interfere with Ab-mediated control of spirochete load in the tissues. These findings demonstrate that the pathogenesis, but not the protective immune response, in Lyme arthritis is dependent on the alpha4beta1-mediated influx of inflammatory cells. The onset of inflammation can be successfully targeted by treatment with S18407.