Although molecular defects in Th cell regulation are thought to contribute to the severity of symptoms in chronic asthma, Lyn, a Src family kinase implicated in allergic inflammation, is not expressed in T cells. Thus, the etiology of the asthma-like syndrome that develops in Lyn−/− mice is unknown. Beavitt et al. (p. 1867 ) found increased numbers of eosinophils, activated macrophages, and activated CD4+ T cells in BAL from Lyn−/− mice immunized and challenged with OVA compared with wild-type controls. Extensive peribronchial and parenchymal inflammation with longer-lasting eosinophilia and reduced apoptosis was seen in the Lyn−/− animals by histological examination. Activated thoracic-draining lymph node CD4+ T cells from Lyn−/− mice produced higher levels of IL-4 and IL-5, and bystander cells produced higher levels of IFN-γ, than controls. Lyn−/− dendritic cells (DC) had a more immature phenotype, and Ag-pulsed Lyn−/− DC exhibited less phosphorylation of paired Ig-like receptor B; LPS stimulation induced less IL-12 production and less phosphorylation of SH2-domain-containing inositol 5-phosphatase-1 compared with controls. Naive Lyn+/+ mice adoptively transferred with OVA-pulsed Lyn−/− DC had greater mucosal inflammation and exaggerated cytokine production than recipients of OVA-pulsed Lyn+/+ DC. The authors propose that DC are central to the critical role played by Lyn in this mouse model of asthma and suggest that Lyn negatively regulates Th2 mucosal inflammatory responses to Ag delivered by aerosol.

Many cytokines and transcription factors influencing CD4+ T cell differentiation are known, but no Th1-specific cell surface molecules of defined function have been reported. Dardalhon et al. (p. 1558 ) found that one of 20,000 mAbs produced in rats against established mouse Th1 cells bound Th1, but not Th2 or Th0, cells. The Th1-specific molecule was identified as CD226 by expression cloning and DNA sequencing. Flow cytometry showed that CD226 was expressed on nearly all naive CD8+ T cells and on ∼40% of unactivated CD4+ T cells. Its expression was up-regulated on activated CD4+ T cells. Under in vitro polarizing conditions, CD226 was expressed at high levels on Th1 cells but was down-regulated on Th2 cells. SJL/J mice immunized with a myelin peptide and treated with either PBS or rat IgG developed Th1 spleen cells that proliferated in vitro in response to peptide and stained with peptide tetramers. The proliferative response was reduced in spleen cells from immunized mice treated with anti-CD226 mAb. Spleen and lymph node cells from the mAb-treated mice had reduced IFN-γ, and increased IL-2, IL-4, and IL-10, secretion; maximal inhibition of CD4+ T cell proliferation occurred using both T cells and CD11b+ cells from mAb-treated animals vs controls. Anti-CD226 mAb treatment delayed disease onset and reduced severity of experimental autoimmune encephalomyelitis induced in RAG-2−/− SJL/J mice by transfer of activated myelin peptide-specific Th1 cells compared with controls. The data demonstrate that CD226 is up-regulated on activated Th1 mouse cells and plays a costimulatory role in the effector phase of a Th1-driven disease model.

Although type 1 diabetes incidence is almost eliminated in NOD mice whose insulin-dependent diabetes (Idd) loci have been replaced by resistant alleles from nondiabetic strains of mice, the impact of those loci on islet Ag-specific CD8+ T cells is unknown. Martinez et al. (p. 1677 ) adoptively transferred purified CD8+ T cells from several strains of mice transgenic for a TCR specific for influenza virus hemagglutinin (HA) into syngeneic hosts expressing an HA transgene in their pancreatic islets (InsHA). Only transferred cells on a NOD background proliferated and increased in peripheral and pancreatic lymph nodes of NOD recipients; proliferation did not occur in InsHA-negative NOD mice. Both Idd3/5-InsHA and Idd9-InsHA mice were resistant to HA peptide-induced diabetes and had poor HA-specific CD8+ T cell responses. The Idd3/5-InsHA mice had less accumulation of the transferred CD8+ T cells, or adoptively transferred CD4+ T cells specific for islet Ags, in pancreatic lymph nodes compared with NOD-InsHA or Idd9-InsHA mice. Restoration of HA-TCR transgenic cell proliferation was accomplished by anti-CD25 mAb treatment of Idd3/5-InsHA recipients before transfer of congenic CD4+ T cells specific for islet Ags. Depletion of CD25+ T cells from purified recipient CD4+ T cells before co-injection with CD4+ T cells specific for HA also restored proliferation. The data indicate that resistance to type 1 diabetes is mediated by pancreatic lymph node CD4+CD25+ T cells in Idd3/5 transgenic mice but by a different mechanism in Idd9 transgenic mice.

Immature dendritic cells (DC) are activated and mature following recognition of abnormal molecules presented by tumors. However, it is not known what factors, produced by malignant or premalignant cells, influence DC differentiation. Carlos et al. (p. 1628 ) used highly purified tumor MUC1mucin from ascitic fluid of cancer patients to induce pertussis toxin-sensitive migration of immature DC generated from human peripheral blood monocytes. Of two well-characterized rMUC1 molecules, the one with less glycosylation and sialylation had greater chemotactic activity for immature DC. Use of a synthetic unglycosylated MUC1 peptide demonstrated that chemotactic activity resided in the tandem repeat region of MUC1; chemotactic activity was eliminated by addition of a single terminal sugar moiety to the peptide or an Ab specific for an epitope in the tandem repeat sequence. Treatment of myeloid DC, isolated from PBMCs of normal donors, with LPS or the nonchemotactic (glycosylated), but not with chemotactic (unglycosylated), forms of MUC1 resulted in up-regulation of maturation markers. Whereas LPS-treated DC induced a Th1 phenotype in CD4+ T cells, DC treated with glycosylated tumor MUC1 did not. The authors conclude that tumor cell hypoglycosylated MUC1 is chemotactic for circulating immature DC, but the DC are subverted by highly sialylated MUC1 to a state of partial maturation incapable of inducing full Th1 differentiation.

Lipid Ags from microbial pathogens are loaded onto CD1 proteins of APCs for presentation to T cells. However, it is not known whether bacteria or their cell wall lipids play an active role in regulating CD1 expression and/or function. Roura-Mir et al. (p. 1758 ) found by flow cytometry and confocal microscopy that GFP-labeled Mycobacterium tuberculosis infection of human monocytes induced high levels of CD1a, CD1b, and CD1c (group 1), but not CD1d (group 2), expression on infected and bystander cells within 2 days. Organic soluble lipids extracted from M. tuberculosis induced group 1 CD1 expression comparable to that induced by live bacteria. Cells with enhanced group 1 CD1 expression had surface markers of immature myeloid dendritic cells. CD1a, CD1b, and CD1c mRNAs increased 100- to 3000-fold in monocytes cultured with M. tuberculosis lipids before appearance of the encoded proteins at the cell surface; levels of CD1d mRNA and surface protein decreased during the first day of lipid treatment. Activation of lipid Ag-specific T cells occurred in lipid Ag-pulsed monocytes that had been incubated with M. tuberculosis total lipids for 3 days but not in lipid Ag-pulsed untreated monocytes. Lipid mixtures from Staphylococcus aureus and four species of mycobacteria stimulated high level group 1 CD1 expression; the activity purified with two M. tuberculosis polar phospholipids known to agonize TLR-2. Anti-TLR-2 Ab significantly inhibited CD1 induction by M. tuberculosis lipids. The data indicate that some M. tuberculosis lipid Ags activate TCRs, whereas others serve as adjuvants to stimulate TLR-2 to induce group 1 CD1 expression and Ag presentation in APCs.

Activation of neutrophils induces transcription of mediators, most with promoters containing κB or κB-like motifs. Resting neutrophils constitutively express NF-κB proteins and their IκB-α inhibitor in their nuclei. Ear et al. (p. 1834 ) detected the α, β, and γ subunits of the IκB kinase (IKK) signalosome in cytoplasm and nuclei of resting human neutrophils but only in PBMC cytoplasm. Both nuclear and cytoplasmic levels of IKKα diminished within 10 min of LPS or TNF-α stimulation of neutrophils but were restored within 2–3 h. IKKα loss was partially inhibited by a proteasome inhibitor; its reappearance was prevented by cycloheximide. LPS and TNF-α stimulation induced phosphorylation of IKKβ, IKKγ, IκB-α, and the NF-κB subunit RelA in both nucleus and cytoplasm. Only IκB-α and RelA accumulated in the nucleus in cells treated with an inhibitor of nuclear export before stimulation. Cyclopentenone prostaglandins were the only kinase inhibitors tested that prevented LPS or TNF-α stimulation-induced IKKβ phosphorylation, IκB-α degradation, and IKKα loss, and reduced IKKγ and RelA phosphorylation. Detection of IKKα, IKKβ, and IKKγ on immunoblots of sonicated nuclei from activated neutrophils suggested IKK association with chromatin. The data demonstrate that the main events of NF-κB pathway activation occur in the nucleus as well as the cytoplasm of human neutrophils and are accompanied by transient loss of IKKα in both locations.

Gram-negative bacteria contain peptidoglycans and LPS, an inducer of bone resorption in inflammatory diseases. Muramyl dipeptide (MDP), the minimal essential structural unit with immunoadjuvant activity of peptidoglycans, enhances many LPS activities, but its involvement in bone resorption is unknown. Yang et al. (p. 1956 ) found that MDP enhanced osteoclast formation induced by LPS, IL-1α, or TNF-α in cocultures of mouse hemopoietic osteoclast progenitors and primary osteoblasts, but MDP alone had no effect. MDP enhanced LPS-induced increases in receptor activator of NF-κB ligand (RANKL) mRNA and ERK1/2 phosphorylation and a TNF-α-induced increase in RANKL expression in osteoblasts. Osteoclast formation was induced by IL-1α, but not by LPS, in cocultures of wild-type osteoblasts and hemopoietic cells from wild-type mice and by TNF-α in cocultures of wild-type osteoblasts and hemopoietic cells from mice deficient in Toll/IL-1R domain-containing adapter proteins. MDP enhanced both activities as well as TNF-α-induced RANKL mRNA expression in the latter cocultures. LPS stimulated expression of Nod2, an intracellular sensor of MDP, in osteoblasts from wild-type mice but not in those from mice deficient for either TLR4 or MyD88; Nod2 mRNA was induced in MyD88-deficient osteoblasts by TNF-α. Small interference RNAs for Nod1 or Nod2 inhibited MDP-induced up-regulation of RANKL mRNA in osteoblasts. The authors propose that enhanced osteoclast formation induced by LPS or inflammatory cytokines occurs through MDP-induced, Nod2-mediated up-regulation of RANKL expression in osteoblasts.

Summaries written by Dorothy L. Buchhagen, Ph.D.