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Phagocytosing Neisseria
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Neutrophil Responses to fMLP
Interaction of bacterial or mitochondrial N-formylated peptides with receptors on neutrophils stimulates activation of a variety of membrane phospholipids and intracellular kinases involved in signal transduction. However, it is not known if src family kinases are part of the signaling pathway. Fumagalli et al. (p. 3874 ) measured reduced superoxide anion production, transmigration through 1-µm pores, F-actin polymerization, and tyrosine phosphorylation of several cellular proteins in fMLP-treated neutrophils from mice deficient for hemopoietic cell kinase (hck) plus Gardner-Rasheed feline sarcoma viral oncogene homologue (fgr) compared with wild-type cells. Decreased phosphorylations of JNK and the guanine nucleotide exchange factor Vav1 also were noted, and no phosphorylation of two Rac target p21-activated kinases occurred in resting and fMLP-exposed hck–/–fgr–/– mouse cells compared with wild-type controls. A src family kinase inhibitor had effects on resting or fMLP-stimulated wild-type mouse or {ititexf}human neutrophils similar to those seen in hck–/–fgr–/– mouse cells. Akt phosphorylation and calcium mobilization were unaffected in mutant mouse cells or human cells treated with the inhibitor after fMLP stimulation. The authors conclude that src family kinases hck and fgr play an essential role in NADPH oxidase activation by fMLP-stimulated neutrophils via Vav1 phosphorylation and activation of p21-activated kinases.
Controlling Bacteremia
Bacteremia, or high blood levels of bacteria, is a leading cause of death from microbial infections; rapid clearance of the bacteria increases survival. Alugupalli et al. (p. 3740 ) studied various strains of mutant mice infected with Borrelia hermsii, the causative agent of relapsing fever, to determine factors critical to bacteria clearance. Although infected mice deficient in Bruton’s tyrosine kinase (Btk) and B1 cells were able to generate the B. hermsii-specific IgM Abs absolutely required to resolve the infection in a T cell-independent manner, their relapsing episodes of bacteremia were more severe and their recovery more prolonged than wild-type controls. Mice deficient in MyD88, but not mice lacking any of the other three TLR adaptor proteins, and TLR2–/– mice had severely delayed and reduced production of anti-B. hermsii IgM Abs compared with infected wild-type animals. IgM Ab responses also were diminished and delayed in infected TLR1–/–, TLR4–/–, and CD14–/– mice. Mice lacking Btk plus MyD88 had populations of B1a, B1b, and marginal zone B subsets of IgM-secreting mature B cells lower than wild-type but equivalent to Btk–/– mice. However, Btk–/–MyD88–/– mice were completely unable to produce IgM Abs to clear B. hermsii infections. The interpretation of the data is that clearance of B. hermsii bacteremia requires both Btk and MyD88 to generate IgM Abs in an atypical T cell-independent TLR-driven B cell response.
Allelic Variant of Fc
RI
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RI expressed on myeloid lineage cells. FcRI is often not paired with FcR 
-chain, but the function of the unpaired receptor is not known. Wu et al. (p. 3688
) found a common single nucleotide polymorphism resulting in replacement of a serine with a glycine within the cytoplasmic domain of human FcRI. Stimulated rat mast cells transfected with the glycine variant were more efficient at mediating intracellular calcium release, induced more degranulation, and had greater IL-6 and TNF- release than cells transfected with the serine variant. Cytokine release occurred in stimulated mouse cells transfected with the glycine, but not the serine, containing FcRI that also had a transmembrane mutation preventing -chain association. Interaction of FcRI with the Src-family member Lyn was shown by in vitro binding of Lyn to an FcRI cytoplasmic tail fragment, by anti-Lyn Ab recognition of Lyn in mouse cell proteins bound to affinity columns of anti-FcRI mAb, and by anti-FcRI Ab immunoblot detection of FcRI in anti-Lyn Ab immunoprecipitates from cell lysates. Lyn coimmunoprecipitated with the glycine, but not the serine, FcRI mutant containing the transmembrane mutation. Neutrophils from human donors homozygous for the glycine allele produced more IL-6 after cross-linking with human IgA compared with those homozygous for the serine allele. The glycine allele was found at higher frequency in African Americans compared with European-American controls and was enriched in patients from both groups with systemic lupus erythematosus. The authors propose that the naturally occurring glycine allele of FcRI increases receptor signaling by direct interaction with Lyn and predisposes individuals to systemic lupus erythematosus. Tether, Roll, Arrest
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(SDF-1). Optimal PBL tethering to VCAM-1 adsorbed to slides occurred at low density of ligand under low flow conditions; firm adhesion (arrest) was preferred over rolling at high-density VCAM-1. Computer-assisted image processing techniques and high-speed video microscopy established at the millisecond level that unstimulated PBL tethering to low site density VCAM-1 had a bimodal pattern with either transient or stable interactions; coimmobilization of SDF-1 with VCAM-1 significantly increased stable, and reduced transient, tethers. Interactions of PBL with VCAM-1 at high site density were nearly twice as stable as interactions with VCAM-1 at low site density. Coimmobilized SDF-1 increased the low site density stable tethers and increased the number of arrested vs rolling PBLs; pertussis toxin blocked the SDF-1-induced increase. Titration of an anti-VLA-4 4 subunit mAb increased firm adhesion with VCAM-1 relative to rolling, whereas a small peptide that binds selectively to high-affinity VLA-4 increased rolling over firmly adherent interactions. The authors propose that SDF-1 induction of high-affinity VLA-4 is critical for arrest of tethered PBL after a brief rolling interaction. Apoptosis of IRF-2–/– Macrophages
Vogel and colleagues have shown that mice lacking IFN regulatory factor 2 (IRF-2) survive LPS challenge better than wild-type animals and have higher numbers of apoptotic Kupffer cells in their livers. Cuesta et al. (p. 3602
) in the Vogel laboratory measured more apoptosis in IRF-2–/– vs wild-type macrophages at the basal level and after exposure to two different apoptosis inducers. Database analyses identified putative STAT binding sites within most of the genes shown by hybridization to DNA microarrays to be down-regulated preferentially in livers of IRF-2–/– animals treated with LPS or saline. Protein and mRNA levels were lower for STAT3
but higher for STAT3 in IRF-2–/– macrophages with or without stimulation; mRNA for caspase-1 was up-regulated. However, STAT3-, STAT1-, and p38 MAPK-activated phosphorylation levels were constitutively higher in unstimulated IRF-2–/– cells and increased to a greater extent after stimulation than in wild-type cells. Cell death was decreased in IRF-2–/–, but not wild-type, cells pretreated with a STAT3 inhibitory peptide, a caspase-1 inhibitor, or a p38 MAPK inhibitor before apoptosis induction. A novel IRF binding site in the promoter of the CASP1 gene was identified by computer analyses and shown by EMSA to bind to a nuclear complex containing IRF-1 plus IRF-2 from stimulated or unstimulated wild-type cells; binding of the complex was enhanced in IRF-2–/– cells. The data suggest that IRF-2 negatively regulates mouse macrophage apoptosis via STAT1/STAT3 inhibition of caspase-1 expression.
STAT4 Is Pivotal in CIA
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, IL-6, and TNF-. Although IFN-, but not IL-6 or TNF-, production from mutant cells was decreased at day 10 after immunization, collagen induced equal proliferation of mutant and wild-type T cells. STAT4 protein had a bimodal increase in expression at 27 and 37 days postimmunization in splenocytes from wild-type mice. Transfection with STAT4 antisense oligonucleotides targeted to the translation start site decreased STAT4 mRNA and protein as detected by RT-PCR and EMSA, respectively. Collagen-immunized mice injected i.v. with the STAT4 antisense oligonucleotides on days 26 and 33 after CIA induction had significantly reduced incidence and severity of arthritis compared with controls. The authors demonstrate the centrality of transcription factor STAT4 in CIA pathogenesis and suggest that its primary influence is on IFN- production that drives inflammation and recruitment of effector cells. Summaries written by Dorothy L. Buchhagen, Ph.D.
Related articles in The JI:
Differentiates Latent Tuberculosis Infection from Disease
Triggers Rapid VLA-4 Affinity Increases to Stabilize Lymphocyte Tethers on VCAM-1 and Subsequently Initiate Firm Adhesion
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