Shoichi Takano

CRTH2, an orphan receptor of T‐helper‐2‐cells, is expressed on basophils and eosinophils and responds to mast cell‐derived factor(s)

We have recently cloned a putative chemoattractant receptor, named CRTH2, which is preferentially expressed on human T‐helper‐ (Th) 2 but not Th1 cells. In this study, we demonstrated that CRTH2 is also highly expressed on peripheral blood basophils and eosinophils. Our search for a CRTH2 ligand identified mast cells as the possible producers of a ligand. When stimulated with an anti‐FcϵR1 antibody, cord blood‐derived mast cells secreted factor(s) that induced Ca2+ mobilization in CRTH2‐expressing K562 cells but not in mock transfected cells. These findings implied the involvement of CRTH2 in mast cell‐mediated immune responses such as allergic reactions.

Cutting Edge: Differential Production of Prostaglandin D2 by Human Helper T Cell Subsets

Several effector molecules, including cytokines, are differentially produced by Th1 and Th2 cells. We used a gene expression screen method to identify a gene encoding hematopoietic PG D synthase (hPGDS) which was preferentially expressed in human Th2 but not Th1 clones. Studies with anti-hPGDS mAbs confirmed the Th2-dominated expression of hPGDS protein. Upon stimulation with anti-CD3 plus anti-CD28 mAbs, coordinated cyclooxygenase-2 expression and PGD2 production were induced in Th2 lines. hPGDS expression was also observed in a small population (<1.0%) of peripheral blood CD4+ lymphocytes from healthy adults. Most hPGDS-expressing CD4+ lymphocytes showed a typical Th2-type cytokine pattern. Our results suggest that, at the sites of Ag presentation, at least part of the Th2 cell population produces PGD2, which may be involved in various aspects of Th2-related immune responses similar to mast cells.

Cutting Edge: Agonistic Effect of Indomethacin on a Prostaglandin D2 Receptor, CRTH2

Indomethacin is a widely used nonsteroidal anti-inflammatory drug and is generally known to exhibit its multiple biological functions by inhibiting cyclooxygenases or activating peroxisome proliferator-activated receptors. In this study, we present evidence demonstrating that the novel PGD2 receptor chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) is another functional target for indomethacin. Indomethacin induced Ca2+ mobilization in CRTH2-transfected K562 cells at submicromolar concentrations (approximate EC50, 50 nM) in a Gαi-dependent manner as PGD2 did. Other nonsteroidal anti-inflammatory drugs (aspirin, sulindac, diclofenac, and acemetacin) had no such effect even at micromolar concentrations. In chemotaxis assay, three CRTH2-expressing cell types, Th2 cells, eosinophils, and basophils, were all significantly attracted by indomethacin (EC50, 50–500 nM) as well as by PGD2 (EC50, 2–20 nM), and the effects of indomethacin were blocked by anti-CRTH2 mAb. These results suggest the involvement of CRTH2 in mediating some of therapeutic and/or unwanted side effects of indomethacin, independently of cyclooxygenases and peroxisome proliferator-activated receptors.

Granulysin in human serum as a marker of cell‐mediated immunity

Granulysin is a cytolytic granule protein of natural killer (NK) cells and cytotoxic T lymphocytes (CTL) with a broad range of antimicrobial and tumoricidal activities. Two molecular forms of granulysin, the 15‐kDa precursor and 9‐kDa mature form, are produced in these cells. In this study, we developed monoclonal antibodies against granulysin and found that the 15‐kDa granulysin is spontaneously secreted by peripheral blood NK and T cells via a non‐granule exocytotic pathway. When NK cells killed the target cells, the released granulysin levels in culture supernatants significantly increased through the granule exocytosis. The granulysin protein was found in the sera of healthy individuals at an average concentration of 3.7±3.2 ng/ml (age 0–99 years, n=244). The serum levels of granulysin were transiently highly elevated among patients with acute viral infections. In addition, the serum granulysin levels in patients with severe immunodeficiency treated bycell therapy fluctuated proportionately to the improvement of other immunological parameters. Our results suggest that granulysin is well associated with diverse activities of NK cells and CTL in physiological and pathological settings and could be a useful novel serum marker to evaluate the overall status of host cellular immunity.

Differential expression of granulysin and perforin by NK cells in cancer patients and correlation of impaired granulysin expression with progression of cancer.

Abstract. Granulysin has been identified as an effector molecule co-localized with perforin in the cytotoxic granules of cytotoxic T lymphocytes and natural killer (NK) cells, and has been reported to kill intracellular pathogens in infected cells in the presence of perforin and to induce a cytotoxic effect against tumor cells. The aim of the present study was to elucidate whether intracellular expression of granulysin and perforin by NK cells might be associated with progression of cancer. Flow cytometric analysis demonstrated high levels of perforin and granulysin expression by CD3– CD16+ cells in healthy controls. In contrast, cancer patients exhibited significantly decreased levels of granulysin expression (P<0.005), despite having equally high levels of perforin expression in comparison with healthy controls. The tumor-free patients expressed granulysin at levels similar to healthy controls, while the progressive tumor-bearing patients expressed remarkably lower levels of granulysin compared to healthy controls (P<0.0001). Similarly, patients with an advanced performance status had significantly fewer granulysin-positive NK cells than healthy controls. Meanwhile, a considerable number of the tumor-bearing patients showed a decrease in the number of circulating NK cells, and a correlation between impaired granulysin expression and reduced circulating NK cells was observed. These findings suggest that the tumor-bearing patients with impaired granulysin expression were in an immunosuppressive state. In conclusion, impaired expression of granulysin by NK cells correlates with progression of cancer, and determination of granulysin expression might prove informative for assessing the immunological condition of cancer patients.

Low Molecular Weight Immunosuppressive Factors Found in Elevated Amounts in Cancer Ascitic Fluids of Mice 2. 1-Methyladenosine Isolated from Cancer Ascitic Fluids Enhances Listeria Infection in Mice

The low molecular weight fraction (mol wt less than 1,000) of Ehrlich cancer ascitic fluid has been known to enhance Listeria infection in mice. Chemical characterization of the entities in this fraction revealed four purine and pyrimidine analogues, i.e. uric acid, uracil, pseudouridine and 1-methyladenosine (m1Ado). When the effect of each of these components was studied on Listeria infection in mice, only m1Ado markedly enhanced the infection and killed the mice within a short period. The optimal enhancement was obtained when m1Ado was given intravenously to mice 3-6 days before the infection at a concentration of between 1 and 100 micrograms/mouse. On the other hand, uric acid, uracil and pseudouridine failed to show such an enhancing effect. m1Ado inhibited macrophage accumulation in the peritoneal cavity of mice after an intraperitoneal injection of phytohemagglutinin. Although m1Ado did not show any inhibitory effect on the phagocytic and bactericidal activities of macrophages in vitro, peritoneal macrophages obtained from mice which received m1Ado 3 days ahead revealed impaired bactericidal activity, suggesting the migration of different cell populations from the bone marrow of m1Ado-receiving mice. The results may suggest that m1Ado is a major factor in tumor ascites causing, in small doses, an impairment of macrophage functioning as can be detected in tumor-bearing hosts.

The use of BRM-activated killer cells in adoptive immunotherapy : A pilot study with nine advanced cancer patients

Adoptive immunotherapy using MHC-nonrestricted-lymphocytes, peripheral blood γδT cells and NK cells was devised. Peripheral blood mononuclear cells (3 x 107) were selected by immobilization to anti-CD3 monoclonal antibody for 4 days and cultured for 2 weeks in the presence of IL-2. Thereafter they were reactivated by 500 U/ml of IFN-α and 1000 U/ml of IL-2 for 1 hour. Enhancement of NK and LAK activities was confirmed. Peripheral blood γδT cells proliferated in response to immobilized anti-CD3 antibody (3% to 30%). Approximately 6 x 109 BRM-activated killer (BAK) cells composed of CD56+ γδT cells and CD56+ NK cells, were dispensed to cancer patients via intravenous drip infusion. Nine patients were treated with BAK cells every 2 weeks or every month on an outpatient basis. During the course of adoptive immunotherapy, the crossed affinity immunoelectrophoresis (CAIE) pattern of serum immunosuppressive acidic protein (IAP) was analysed. Both the production and glycosylation pattern of IAP is changed in response to tumor enlargement and may therefore act as a marker of the disease progression. During the course of BAK therapy, the glycosylation IAP pattern of 6 patients changed from tumor (T) to normal (N). In addition, the performance status of all patients was maintained at 90–100% of the Karnofsky scale and any side effects including fever were not observed during treatments with BAK cells. Moreover, the overall quality of life (QOL) of the patients, scored at the Face scale was favorable. In addition, blood levels of activated γδT cells producing IFN-γ were assayed as an indication marker of BAK therapy. The normal range of IFN-γ producing γδT cells comprised 6.9 ± 0.9% of peripheral blood mononuclear cells (PBMC), according to a single cell FACScan analyses of PBMCs derived from normal individuals. IFN-γ producing γδT cells of Patients No. 8 and 9, who received extensive chemotherapy before initiation of BAK therapy, comprised only 0.2% and 2% of PBMC, respectively. These patients died 3 and 6 months after beginning BAK therapy. Peripheral blood γδT cells of Patients Nos. 1–7 proliferated in response to immobilized anti-CD3 antibody and the frequency of IFN-γ producing γδT cells in PBMC preparation of these patients were over 3% before initiation of BAK therapy. Since our data show a positive correlation between survival time and initial γδT cell counts, a low frequency of these cells may contraindicate BAK therapy.

Low Molecular Weight Immunosuppressive Factors Found in Elevated Amounts in Cancer Ascitic Fluids of Mice 1. Isolation, Identification and Immunosuppressive Effects of Uric Acid and Uracil

A definite increase in two low molecular weight factors, G10-2 and G10-3 was found in Ehrlich ascitic fluids, parallel to tumor growth. The isolation and identification of the two factors were attempted through gel filtration and reversed phase column chromatography, using ascitic fluids obtained 13 days after intraperitoneal implantation of Ehrlich tumor cells. As a result, two highly purified factors were observed upon examination by high performance liquid chromatography. Additional analytical data, collected by UV spectrum, NMR spectrum and mass analysis, allowed us to identify G10-2 as uric acid and G10-3 as uracil. Detailed immunological analysis of uric acid and uracil revealed that the augmenting activities of mouse and human NK cells by mouse IFN alpha/beta or human rIFN alpha A/D were impaired in the presence of either compound at concentrations of 0.07 mM, the concentration detectable in the ascitic fluid of tumor bearing mice.

Isolation of a factor from cancer ascitic fluid increasing susceptibility of mice to Pseudomonas infection.

Abstract A factor which impairs the capacity of normal recipient mice to resist Pseudomonas infection has been isolated from the ascitic fluids of tumor-bearing mice. This factor was dialyzable (molecular weight between 4000 and 10,000), showed potent suppressive activity when as little as 10 μg was given ip and was resistant to heating at 56 for °30 min. The factor in the ascitic fluids was detectable rather late after tumor implantation and the production of the factor in the ascitic fluids was closely related to the tumor growth.