Langerhans cells (LCs) are prominent dendritic cells (DCs) in epithelia, but their role in immunity is poorly defined. To track and discriminate LCs from dermal DCs in vivo, we developed knockin mice expressing enhanced green fluorescent protein (EGFP) under the control of the langerin (CD207) gene. By using vital imaging, we showed that most EGFP(+) LCs were sessile under steady-state conditions, whereas skin inflammation induced LC motility and emigration to lymph nodes (LNs). After skin immunization, dermal DCs arrived in LNs first and colonized areas distinct from slower migrating LCs. LCs reaching LNs under steady-state or inflammatory conditions expressed similar levels of costimulatory molecules. Langerin and EGFP were also expressed on thymic DCs and on blood-derived, CD8alpha(+) DCs from all secondary lymphoid organs. By using a similar knockin strategy involving a diphtheria toxin receptor (DTR) fused to EGFP, we demonstrated that LCs were dispensable for triggering hapten-specific T cell effectors through skin immunization.

The early innate response after Mycobacterium bovis bacille Calmette-Gué rin (BCG) vaccination is poorly characterized but probably decisive for subsequent protective immunity against tuberculosis. Therefore, we vaccinated mice with fluorescent BCG strains in the ear dorsum, as a surrogate of intradermal vaccination in humans. During the first 3 days, we tracked BCG host cells migrating out of the dermis to the auricular draining lymph nodes (ADLNs). Resident skin dendritic cells (DCs) or macrophages did not play a predominant role in early BCG capture and transport to ADLNs. The main BCG host cells rapidly recruited both in the dermis and ADLNs were neutrophils. Fluorescent green or red BCG strains injected into nonoverlapping sites were essentially sheltered by distinct neutrophils in the ADLN capsule, indicating that neutrophils had captured bacilli in peripheral tissue and transported them to the lymphoid organ. Strikingly, we observed BCG-infected neutrophils in the lumen of lymphatic vessels by confocal microscopy on ear dermis. Fluorescencelabeled neutrophils injected into the ears accumulated exclusively into the ipsilateral ADLN capsule after BCG vaccination. IntroductionMycobacterium bovis bacille Calmette-Guérin (BCG) is the only available vaccine against tuberculosis (TB), a major public health problem. Being included in the World Health Organization (WHO) Expanded Program for Immunization, BCG is one of the most widely administered vaccines. It confers high levels of protection against disseminated forms of TB, particularly severe in children, but its efficacy against pulmonary TB in adults is estimated to be only 50% 1 and varies widely among different geographic areas and populations. Thus, more efficient vaccines against TB are urgently needed. There are reasons to believe that such vaccines could be based on BCG. Therefore, a better understanding of the immune response induced by BCG could help in designing better strategies on a rational basis. Today, BCG vaccination is almost exclusively administered intradermally or percutaneously. 2 Early events occurring after BCG vaccination that will have a strong impact on the adaptive immune response are poorly characterized. For example, it is unknown how BCG travels from the injection site to draining lymph nodes (DLNs) and which host cells could be involved in this early process. Mononuclear phagocytes such as epidermal Langerhans cells (LCs), dermal macrophages, and dendritic cells (DCs) are ideally located to capture microorganisms entering skin. Due to their migratory capacity, DCs shuttle pathogens such as HIV 3 or Leishmania major 4 to DLNs. Bacterial dissemination from gut to mesenteric DLNs occurs via infected DCs after ingestion of Salmonella 5 or Listeria. 6 Peripheral tissue DCs are not the only cells at play in bridging innate and acquired immunity to pathogens. Soon after an inflammatory stimulus, blood monocytes are recruited to the injured tissue from which they can migrate via afferent lymph toward DLNs. There, monocytes acquire a DC ...

The mammalian proto-oncoprotein Cbl and its homologues inThis mutant includes the evolutionarily conserved TKB and RING finger domains but lacks the less conserved C-terminal sequences. We conclude that the evolutionarily conserved N terminus of Cbl is sufficient to effect enhancement of EGF-R ubiquitination and down-regulation from the cell surface.

Dendritic cells are professional antigen-presenting cells that initiate primary immunity. Migration from sites of antigen uptake to lymphoid organs is crucial for the generation of immune responses. We investigated the migratory pathways specifically of epidermal Langerhans cells by tracing them from the epidermis to the draining lymph nodes. This was possible with a new monoclonal antibody, directed against murine Langerin/CD207, a type II lectin specific for Langerhans cells. In situ, resident, and activated Langerhans cells express Langerin in the epidermis and on their way through dermal lymphatic vessels. Both emigrated and trypsinization-derived Langerhans cells expressed high levels of Langerin intracellularly but reduced it upon prolonged culture periods. Sizeable numbers of Langerin+ cells were found in skin draining lymph nodes but not in mesenteric nodes. Langerin+ cells localized to the T cells areas and rarely to B cell zones. Numbers of Langerin-expressing cells increased after application of a contact sensitizer. In the steady state, Langerhans cells in the skin-draining nodes expressed maturation markers, such as 2A1 and costimulatory molecules CD86 and CD40. These molecules, CD86 and CD40, were further upregulated upon inflammatory stimuli such as contact sensitization. Thus, the novel anti-Langerin monoclonal antibody permits the unequivocal visualization of migratory Langerhans cells in the lymph nodes for the first time and thereby allows to dissect the relative immunogenic or tolerogenic contributions of Langerhans cells and other types of dendritic cells.

The Cbl proto-oncogene product has emerged as a novel negative regulator of receptor and non-receptor tyrosine kinases. Our previous observations that Cbl overexpression in NIH3T3 cells enhanced the ubiquitination and degradation of the platelet-derived growth factor receptor-␣ (PDGFR␣) and that the expression of oncogenic Cbl mutants up-regulated the PDGFR␣ signaling machinery strongly suggested that Cbl negatively regulates PDGFR␣ signaling. Here, we show that, similar to PDGFR␣, selective stimulation of PDGFR␤ induces Cbl phosphorylation, and its physical association with the receptor. Overexpression of wild type Cbl in NIH3T3 cells led to an enhancement of the ligand-dependent ubiquitination and subsequent degradation of the PDGFR␤, as observed with PDGFR␣. We show that Cbl-dependent negative regulation of PDGFR␣ and ␤ results in a reduction of PDGF-induced cell proliferation and protection against apoptosis. A point mutation (G306E) that inactivates the tyrosine kinase binding domain in the N-terminal transforming region of Cbl compromised the PDGF-inducible tyrosine phosphorylation of Cbl although this mutant could still associate with the PDGFR. More importantly, the G306E mutation abrogated the ability of Cbl to enhance the ligand-induced ubiquitination and degradation of the PDGFR and to inhibit the PDGF-dependent cell proliferation and protection from apoptosis. These results demonstrate that Cbl can negatively regulate PDGFR-dependent biological responses and that this function requires the conserved tyrosine kinase binding domain of Cbl.

Donor-specific (DST) or nonspecific blood transfusions administered before transplantation can enhance survival of vascularized allografts both in humans and animals but the immunological mechanisms of this effect remain unclear. We have analyzed the expression and the role of endogenous TGF-␤ 1 in a model of heart allograft tolerance, induced by pregraft DST in adult rats. We reported previously that this tolerance occurs despite a strong infiltration of leukocytes into the graft that are unable to produce both Th1-and Th2-related cytokines in vivo. Allografts from DST-treated rats express high levels of TGF-␤ 1 mRNA and active protein. This phenomenon is correlated with the rapid infiltration of leukocytes producing high amounts of TGF-␤ 1. TGF-␤ 1-producing cells are virtually absent among early infiltrating cells in rejected grafts but are found at a later time point. The induction of allograft tolerance in vivo is abrogated by administration of neutralizing anti-TGF-␤ mAb. Moreover, overexpression of active TGF-␤ 1 in heart allografts using a recombinant adenovirus leads to prolonged graft survival in unmodified recipients. Taken together, our results identify TGF-␤ as a critical cytokine involved in the suppression of allograft rejection induced by DST and suggest that TGF-␤ -producing regulatory cells are also involved in allograft tolerance. ( J. Clin. Invest. 1998.

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Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine and counterregulator of glucocorticoids, is a potential therapeutic target. MIF is markedly different from other cytokines because it is constitutively expressed, stored in the cytoplasm, and present in the circulation of healthy subjects. Thus, the concept of targeting MIF for therapeutic intervention is challenging because of the need to neutralize a ubiquitous protein. In this article, we report that MIF occurs in two redox-dependent conformational isoforms. We show that one of the two isoforms of MIF, that is, oxidized MIF (oxMIF), is specifically recognized by three mAbs directed against MIF. Surprisingly, oxMIF is selectively expressed in the plasma and on the cell surface of immune cells of patients with different inflammatory diseases. In patients with acute infections or chronic inflammation, oxMIF expression correlated with inflammatory flare-ups. In addition, anti-oxMIF mAbs alleviated disease severity in mouse models of acute and chronic enterocolitis and improved, in synergy with glucocorticoids, renal function in a rat model of crescentic glomerulonephritis. We conclude that oxMIF represents the disease-related isoform of MIF; oxMIF is therefore a new diagnostic marker for inflammation and a relevant target for anti-inflammatory therapy.