Shiro Kanegasaki

Role of CC chemokine receptor 2 in bone marrow cells in the recruitment of macrophages into obese adipose tissue.

The MCP-1 (monocyte chemoattractant protein-1)/CCR2 (CC motif chemokine receptor-2) pathway may play a role in macrophage infiltration into obese adipose tissue. Here we investigated the role of CCR2 in the recruitment of bone marrow-derived macrophages into obese adipose tissue in vitro and in vivo. Using the TAXIScan device, which can measure quantitatively the directionality and velocity of cell migration at time lapse intervals in vitro, we demonstrated that bone marrow cells (BMCs) from wild type mice migrate directly toward MCP-1 or culture medium conditioned by adipose tissue explants of genetically obese ob/ob mice, which are efficiently suppressed by pharmacological blockade of CCR2 signaling. The number of F4/80-positive macrophages was reduced in the adipose tissue from high fat diet-fed obese KKAy or ob/ob mice treated with a CCR2 antagonist propagermanium relative to vehicle-treated groups. We also found that the number of macrophages is reduced in the adipose tissue from ob/ob mice reconstituted with CCR2-/- BMCs (ob/ob + CCR2-/- BMCs) relative to those with CCR2+/+ BMCs (ob/ob + CCR2+/+ BMCs). Expression of mRNAs for CD11c and TLR4 (Toll-like receptor 4) markers of proinflammatory M1 macrophages was also decreased in the adipose tissue from ob/ob + CCR2-/- BMCs relative to ob/ob + CCR2+/+ BMCs, whereas mannose receptor and CD163, markers of anti-inflammatory M2 macrophages, were unchanged. This study provides in vivo and in vitro evidence that CCR2 in bone marrow cells plays an important role in the recruitment of macrophages into obese adipose tissue.

Lungs of patients with idiopathic pulmonary alveolar proteinosis express a factor which neutralizes granulocyte‐macrophage colony stimulating factor

Mice deficient in granulocyte‐macrophage colony stimulating factor (GM‐CSF) develop pulmonary alveolar proteinosis (PAP). We found that bronchoalveolar lavage fluid (BALF) from 11 patients with idiopathic pulmonary alveolar proteinosis (IPAP) suppressed the growth of peripheral blood monocytes and TF‐1 cells, a cell line dependent on either GM‐CSF or interleukin‐3 (IL‐3). The inhibitory effect of PAP‐BALF occurred only when TF‐1 cells were cultured with GM‐CSF but not when cultured with IL‐3, suggesting that PAP‐BALF contains a factor that specifically interferes with GM‐CSF function. 125I‐GM‐CSF binding to TF‐1 cells was prevented in the presence of BALF from IPAP patients. Furthermore, cross‐linking of 125I‐GM‐CSF to IPAP‐BALF produced two major bands on SDS‐PAGE; these bands were not observed in normal BALF. These data suggest that IPAP is caused by expression of binding factor(s) which inhibit GM‐CSF function in the lung.

Enhancement of Antitumor Radiation Efficacy and Consistent Induction of the Abscopal Effect in Mice by ECI301, an Active Variant of Macrophage Inflammatory Protein-1α

Purpose: We studied whether i.v. administration of a chemokine after local tumor site irradiation could prevent remaining, as well as distant, nonirradiated tumor cell growth by leukocyte recruitment. Experimental Design: Tumors were implanted s.c. in the right or both flanks. After local irradiation at the right flank, ECI301, a human macrophage inflammatory protein-1α variant was injected i.v. Tumor volumes were measured every 3 days after treatment. Results: In Colon26 adenocarcinoma-bearing BALB/c mice, repeated daily administration (over 3-5 consecutive days) of 2 μg per mouse ECI301 after local irradiation of 6 Gy prolonged survival without significant toxicity, and in about half of the treated mice, the tumor was completely eradicated. Three weekly administrations of ECI301 after local irradiation also led to significant, although less effective, antitumor radiation efficacy. ECI301 also inhibited growth of other syngenic tumor grafts, including MethA fibrosarcoma (BALB/c) and Lewis lung carcinoma (C57BL/6). Importantly, tumor growth at the nonirradiated site was inhibited, indicating that ECI301 potentiated the abscopal effect of radiation. This abscopal effect observed in BALB/c and C57BL/6 mice was tumor-type independent. Leukocyte depletion studies suggest that CD8+ and CD4+ lymphocytes and NK1.1 cells were involved. Conclusions: Marked inhibition of tumor growth at the irradiated site, with complete tumor eradication and consistent induction of the abscopal effect, was potentiated by i.v. administration of ECI301. The results of this study may offer a new concept for cancer therapy, namely chemokine administration after local irradiation, leading to development of novel therapeutics for the treatment of advanced metastatic cancer.

Pivotal function for cytoplasmic protein FROUNT in CCR2-mediated monocyte chemotaxis

Ligation of the chemokine receptor CCR2 on monocytes and macrophages with its ligand CCL2 results in activation of the cascade consisting of phosphatidylinositol-3-OH kinase (PI(3)K), the small G protein Rac and lamellipodium protrusion. We show here that a unique clathrin heavy-chain repeat homology protein, FROUNT, directly bound activated CCR2 and formed clusters at the cell front during chemotaxis. Overexpression of FROUNT amplified the chemokine-elicited PI(3)K–Rac–lamellipodium protrusion cascade and subsequent chemotaxis. Blocking FROUNT function by using a truncated mutant or antisense strategy substantially diminished signaling via CCR2. In a mouse peritonitis model, suppression of endogenous FROUNT markedly prevented macrophage infiltration. Thus, FROUNT links activated CCR2 to the PI(3)K–Rac–lamellipodium protrusion cascade and could be a therapeutic target in chronic inflammatory immune diseases associated with macrophage infiltration.

Location of the epitope for 7D5, a monoclonal antibody raised against human flavocytochrome b558, to the extracellular peptide portion of primate gp91phox.

Flavocytochrome b558 is the membrane component of the phagocyte NADPH oxidase, and is a heterodimer composed of gp91phox and p22phox subunits. Human flavocytochrome b558 is recognized by monoclonal antibody 7D5 at an unidentified extracellular domain, although our previous study suggested it might recognize p22phox. 7D5 has proven useful in rapid screening of individuals for X‐linked chronic granulomatous disease by flow‐cytometry. Therefore, we re‐evaluated the location of the 7D5 epitope using gene‐engineered cell lines expressing hybrid flavocytochromes composed of human and murine subunit homologues. The current study demonstrates that the 7D5 recognizes epitope only of primate gp91phox. Flow‐cytometric analyses showed that 7D5 consistently bound to cells expressing human gp91phox. In addition, 7D5 immunoprecipitated the ~58 kDa unglycosylated gp91phox protein from solubilized membrane fractions of tunicamycin‐treated PLB‐985 granulocytes, indicating that glycans were not required for 7D5 binding. Transgenic COS7 cells expressing human gp91phox but not p22phox were recognized by 7D5. These results localized the epitope of 7D5 to an extracellular peptide portion of primate gp91phox and indicate that the antibody will be useful for monitoring the efficiency of gene therapy in patients with flavocytochrome b558‐deficient chronic granulomatous disease and for elucidating structural characteristics of flavocytochrome b558.

Apoptosis under hypercytokinemia is a possible pathogenesis in influenza-associated encephalopathy.

Abstract  Background : Influenza‐associated encephalopathy is reported to be frequent in Japan and East Asia. No evaluating markers except interleukin (IL)‐6 and tumor necrosis factor (TNF)‐α and no likely pathological mechanism for the disease have yet been elucidated.

SMOOTH SPECIFIC PHAGE ADSORPTION: ENDORHAMNOSIDASE ACTIVITY OF TAIL PARTS OF P22

Abstract The tail parts of phage P22 as well as the phage particles cleave the O-antigen of its host bacterium, Salmonella typhimurium . The cleavage is caused by specific breakage of α-rhamnosyl 1–3 galactose linkages. Thus the tail parts of this phage consist of an enzyme, endorhamnosidase. The enzyme was not detected in nonpermissible strain infected with an amber gene 9 mutant of P22. Head without tail parts gains infectivity only after incubation with the tail parts which carry this enzymatic activity.

Pyridine and imidazole reversibly inhibit the respiratory burst in porcine and human neutrophils: evidence for the involvement of cytochrome b558 in the reaction.

Heterocyclic nitrogenous bases, such as pyridine and imidazole which bind to heme-iron in cytochromes, inhibited the respiratory burst in intact neutrophils and NADPH-dependent oxygen consumption in lysed cells. Inhibition was accompanied by a spectral change in reduced cytochrome b558 as judged by low-temperature spectroscopy at 77 K. The position and shape of the alpha-band of the cytochrome were significantly altered upon exposure to pyridine or some other bases. Both inhibition and spectral changes were reversible. The results are consistent with the view that cytochrome b558 is involved in the NADPH oxidase system in neutrophils.

Differential Regulatory Function of Resting and Preactivated Allergen-Specific CD4+CD25+ Regulatory T Cells in Th2-Type Airway Inflammation

Although CD4+CD25+ regulatory T (Treg) cells are known to suppress Th1 cell-mediated immune responses, their effect on Th2-type immune responses remains unclear. In this study we examined the role of Treg cells in Th2-type airway inflammation in mice. Depletion and reconstitution experiments demonstrated that the Treg cells of naive mice effectively suppressed the initiation and development of Th2-driven airway inflammation. Despite effective suppression of Th2-type airway inflammation in naive mice, adoptively transferred, allergen-specific Treg cells were unable to suppress airway inflammation in allergen-presensitized mice. Preactivated allergen-specific Treg cells, however, could suppress airway inflammation even in allergen-presensitized mice by accumulating in the lung, where they reduced the accumulation and proliferation of Th2 cells. Upon activation, allergen-specific Treg cells up-regulated CCR4, exhibited enhanced chemotactic responses to CCR4 ligands, and suppressed the proliferation of and cytokine production by polarized Th2 cells. Collectively, these results demonstrated that Treg cells are capable of suppressing Th2-driven airway inflammation even in allergen-presensitized mice in a manner dependent on their efficient migration into the inflammatory site and their regulation of Th2 cell activation and proliferation.

Chemotherapeutic activity of synthetic antimicrobial peptides: correlation between chemotherapeutic activity and neutrophil-activating activity

The chemotherapeutic activity of three synthetic antibacterial peptides was investigated. KLKLLLLLKLK‐NH2 and its d‐enantiomer showed significant chemotherapeutic activity in MRSA‐infected mice, whereas KLKLLLKLK‐NH2, which showed the highest antibacterial activity among them in vitro, was found to have almost no ability to prevent MRSA infection. These results suggest that the antibacterial activity of peptides assessed in vitro does not necessarily correlate with their chemotherapeutic activity. We found that KLKLLLLLKLK‐NH2 and its d‐enantiomer, but not KLKLLLKLK‐NH2, have the ability to activate human neutrophils to produce superoxide, suggesting that the prevention of MRSA infection by these peptides is not simply due to their direct bactericidal activity but to augmentation of the systemic defense mechanism mediated by neutrophils.