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Endothelin (ET)-1(1-31) is a novel 31-amino acid-length peptide derived from big ET-1 by chymase or other chymotrypsin-type proteases and is a major ET derivative in human neutrophils. In this study, we revealed that ET-1(1-31), but not big ET, exhibited chemotactic activities toward human neutrophils and monocytes as an inflammatory mediator, although the effects were less potent than those of formyl-methionyl-leucyl-phenylalanine or interleukin-8. However, the chemotactic effects of ET-1(1-31) were much greater than those of the 21-amino acid ET-1, ET-1(1-21). Checkerboard analyses revealed that the effects are chemotactic rather than chemokinetic. The effects of ET-1(1-31) are not mediated by interleukin-8 or monocyte chemoattractant protein-1. The chemotactic effects and an increase in intracellular-free Ca2+ caused by ET-1(1-31) were significantly inhibited by BQ123, an ETA receptor antagonist, but not by BQ788, an ETB receptor antagonist, suggesting that ET-1(1-31) mediates chemotaxis through an ETA or ETA-like receptor.

The effect of interleukin-4 (IL-4) on the activation state of human alveolar macrophages (AMs) and blood monocytes induced by lipopolysaccharide (LPS) or recombinant interferon-γ (IFN-γ) was investigated on the basis of their ability to produce superoxide anion (O2-). AMs were obtained from healthy donors by bronchoalveolar lavage, and O2- productions of these cells were assayed by a cytochrome c reduction method after incubation with stimulants for 24 h. AMs produced more O2- than autologous blood monocytes when stimulated with LPS. IL-4 alone had little effect on O2- production by unstimulated AMs but down-regulated O2- production by LPS-stimulated AMs in a dose-dependent manner. IL-4 also suppressed O2- production by AMs induced by the synergistic actions of muramyl dipeptide (norMDP) and IFN-γ. Maximum suppression by IL-4 of O2- production by AMs was observed when IL-4 was added within 1 h after initiation of LPS stimulation. AMs also showed high O2- production when stimulated with IFN-γ alone. In contrast to its suppression of O2- production by LPS-stimulated AMs, IL-4 enhanced O2- production by AMs stimulated with IFN-γ. These data suggest that IL-4 is an important regulator of O2- production by macrophages through different pathways depending on the stimulus.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has stimulatory effects on various monocyte functions. We examined whether all or only some blood monocytes could respond to GM-CSF. Monocytes from peripheral blood of healthy donors were separated by size into five fractions by counter-flow centrifugal elutriation (CCE). The phagocytic activities of monocytes in these fractions depended on the size of the cells. On activation by bacteria-derived stimuli, these fractions showed similar responses of production of monokines such as interleukin-1 (IL-1) and tumor necrosis factor (TNF) and cytotoxicity against allogeneic tumor cells. On treatment of these fractions with optimal concentration of GM-CSF, fractions 3, 4, and 5 showed tumoricidal activity and produced cell-associated IL-1, fraction 3 producing the most, whereas release of IL-1 and TNF in the supernatant was not observed. The cell-associated IL-1 was identified as IL-1α, not IL-1β, by neutralizing tests with antisera against IL-1α and IL-1β. GM-CSF also induced the proliferative and colony-forming responses of medium and large monocytes. These observations suggest that adoptive therapy with macrophage progenitor cells in peripheral blood may be useful in combination with GM-CSF for treatment of monocytopenia after chemotherapy or radiation therapy.

We examined the mechanisms involved in interleukin (IL)-12-mediated suppression of cellular immunity in mice using allogeneic mixed leukocyte reaction (MLR) stimulated by dendritic cells (DCs) in vitro and compared the effect of IL-12 on MLR in mice and humans. Although IL-12 stimulated human MLR, the addition of IL-12 or interferon-γ (IFN-γ) resulted in a dose-dependent suppression of MLR in mice. The treatment with NG-monomethyl-l-arginine (L-NMMA) completely abrogated IL-12- and IFN-γ-mediated suppression of MLR in mice. Furthermore, IL-12 enhanced the alloreactive cytolytic T lymphocyte (CTL) induction in human MLR, whereas the addition of L-NMMA was required to generate alloreactive CTLs in the presence of IL-12 in mice. Nitric oxide (NO) was detected only in mouse MLR. Murine DCs could produce NO, but neither human CD34+ cell- nor monocyte-derived DCs produced a detectable amount of NO. These results suggest that NO produced by DCs might play an important role in IL-12-mediated immune suppression in mice but not in humans.

CC chemokine receptor (CCR)4 is selectively expressed on Th2-type Tcells and has been shown to be responsible for Th2-dominant immuneresponses. In this study, we analyzed the expression of CCR4 in activesystemic lupus erythematosus (SLE) patients by FACS analysis usinganti-human CCR4 monoclonal antibody and determined the clinicalrelevance in this disease. Higher expression of CCR4 was found onperipheral blood CD4+ T lymphocytes of active SLE patients than wasfound with healthy controls and inactive SLE patients. The CCR4expression significantly correlated with the SLE disease activity index(SLEDAI) scores. The expression was dramatically decreased after thecorticosteroid therapy in parallel with a serum level ofdouble-stranded DNA antibody and SLEDAI scores. Moreover, we found thatserum levels of IL-10 were increased in active SLE patients andsignificantly correlated with the CCR4 expression. This study suggeststhat Th2 immune response is predominant in the active state of SLE, andCCR4 may have relevance in regard to the disease course in SLEpatients.

Chymase is a major chymotrypsin-like serine protease expressed in the secretory granules of mast cells in many mammalian species. In this study, we revealed the chemotactic activity of chymase for human mononuclear cells and neutrophils with a 48-well microchemotaxis chamber technique. Human chymase showed the potent chemotactic activity for monocytes and neutrophils dose-dependently in a concentration range from 0.1 to 10 μg/mL, corresponding to about 4–400 μM. The activity was as potent as that of N-formyl-methionyl-leucyl-phenylalanine. Chymase also stimulated cell migration of lymphocytes and purified T cells, but checkerboard analysis revealed that the effect was chemokinetic rather than chemotactic. Inhibition of chymase activities with chymase inhibitors, such as antileukoprotease and Bowman-Birk soybean trypsin inhibitor, significantly inhibited the chemotactic activity of chymase, suggesting that the proteolytic activity of chymase participates in the chemotactic activity. Our results suggest that mast cell chymase acts as a chemoattractant, and may play a role in the accumulation of inflammatory cells in development of the chronic inflammatory responses of allergic and nonallergic diseases. J. Leukoc. Biol. 67: 585–589; 2000.

Dendritic cells (DC) play a key role in the initiation of immune response by stimulating the naive T cells. The fate of DC after the initiation of immune response is not clearly understood. Although there are few reports implicating natural killer (NK) cells in the elimination of DC, killing of DC by LAK cells, and specifically by T cells, has not been studied. In this study, we observed that DC, generated from monocytes, in vitro in the presence of granulocyte-macrophage colony-stimulating factor, interleukin-4 (IL-4), and tumor necrosis factor α were susceptible to cytolysis by lymphokine-activated killer (LAK) cells induced in the presence of IL-2 and IL-15 but not IL-12 alone. However, LAK cells induced by a combination of IL-12 and suboptimal dose of IL-2 were cytotoxic to DC. When purified lymphocytes were activated with IL-2, the CD8+/CD57− fraction (T-LAK), but not the CD8−/CD57+ fraction (NK-LAK) was cytotoxic to autologous DC. However, when unseparated peripheral blood mononuclear cells were used to generate LAK cells, both T-LAK and NK-LAK fractions showed equal cytotoxicity against autologous DC. Monoclonal antibodies against CD54, CD11a, and CD18 significantly inhibited the cytolysis, indicating that the killing involves the engagement of CD54 with its ligands. J. Leukoc. Biol. 65: 764–770; 1999.

FR167653 {1-[7-(4-fluorophenyl)-1, 2, 3, 4-tetrahydro-8 (4-pyridyl) pyrazoro [5-1-c] [1, 2, 4] triazin-2-yl]-2-phenylethanedion sulfate monohydrate} was developed to inhibit proinflammatory cytokine production. However, the effects of FR167653 on prostanoid production are unclear. We investigated the effect of FR167653 on proinflammatory cytokine and prostaglandin (PG) production by lipopolysaccharide (LPS)-stimulated human peripheral blood monocytes and alveolar macrophages (AM) from the same individuals, and compared the effects in monocytes and AM. FR167653 inhibited interleukin-1β and tumor necrosis factor a production. The effect on PGE2 production was assessed by four parameters. FR167653 inhibited PGE2 synthesis and LPS induction of cyclooxygenase activity. Western and Northern blot analyses revealed that LPS induction of cyclooxygenase-2 expression was attenuated by this compound. The suppression in monocytes was greater than that in AM. We concluded that the reduction of LPS-induced PGE2 synthesis by FR167653 was due to inhibition of cyclooxygenase-2 production. These results show that FR167653 may be therapeutically useful for inhibiting production of both inflammatory cytokines and PG production in inflammatory diseases. J. Leukoc. Biol. 65: 80–86; 1999.

The effect of recombinant human IL-4, IL-10, and IL-13 on the chemotaxis and antitumor activity of human blood monocytes induced by monocyte chemotactic and activating factor (MCAF) was examined. MCAF alone did not induce monocyte-mediated cytotoxicity against human melanoma (A375-M) cells whereas it significantly enhanced the cytotoxicity by norMDP-stimulated monocytes. MCAF, unlike IFN-γ, had no priming effect on monocyte activation by norMDP. MCAF acted with norMDP or LPS to enhance the production of both IL-1β and TNF-α. Enhanced cytotoxicity of monocytes stimulated with MCAF plus norMDP was reduced by IL-1 receptor antagonist and anti-TNF-α antibody. IL-4, IL-10, and IL-13 suppressed the generation of antitumor activity and cytokine production (IL-1β and TNF-α) of monocytes stimulated with MCAF plus norMDP or LPS. Chemotaxis of monocytes induced by MCAF was not affected by norMDP or any of the anti-inflammatory cytokines (IL-4, IL-10, and IL-13). Moreover, the pretreatment of monocytes with anti-inflammatory cytokines did not suppress monocyte-chemotaxis. These findings suggest that in vivo recruitment and anti-tumor expression of blood monocytes induced by MCAF may be differently regulated by anti-inflammatory cytokines in vivo. J. Leukoc. Biol. 57: 303–309; 1995.

The effects of purified human interleukin-10 (IL-10) on the expression of antitumor activity of human monocytes and alveolar macrophages (AMs) obtained by centrifugal elutriation and bronchoalveolar lavage, respectively, from the same healthy donors were examined. Monocytes and AMs were incubated for 16 h in medium with lipopolysaccharide (LPS) in the presence or absence of IL-10 or IL-4, and then their tumoricidal activity was assayed by measuring 125I-IUdR release from human melanoma (A375) cells. Addition of IL-10 to cultures of monocytes or AMs with LPS resulted in dose-dependent suppression of their cytotoxicity against A375 cells, the suppression of the activity of monocytes being the higher. IL-10 also suppressed the synergistic effects of interferon-γ and desmethyl muramyldipeptide in activation of monocytes. IL-10 inhibited the early induction phase of monocyte activation but not the effector phase (monocyte-mediated cytotoxicity). IL-10 plus IL-4 inhibited the antitumor activities of AMs and monocytes much more than either IL-10 or IL-4 alone. IL-10 and IL-4 at sub-optimal concentrations also showed synergistic inhibitory effects. These findings suggest that IL-10 may be important in vivo in down-regulating the antitumor activities of monocytes and AMs in the lung by inhibiting their productions of antitumor effector molecules. J. Leukoc. Biol. 55: 437–442; 1994.

The expressions of a membrane form of TNF (m-TNF) by human alveolar macrophages (AM) and autologous blood monocytes from healthy donors were examined. Upon lipopolysaccharide (LPS) stimulation, AM produced 26-kDa TNF-α on their cell surface. We designed a bioassay for measuring m-TNF in which macrophages were fixed with paraformaldehyde after stimulation for 18 h, then m-TNF activity was assessed as cytotoxicity of fixed macrophages on L929 cells. This assay was specific to m-TNF because: 1) no soluble factors were contributed to the cytotoxicity of fixed AM, 2) anti-TNF-α monoclonal antibody completely neutralized m-TNF activity, and 3) m-TNF activity was not altered after low- pH or high-salt treatment. On LPS stimulation, AM produced significant amounts of m-TNF earlier than TNF-α secretion. AM also expressed significant amounts of m-TNF when stimulated with other bacterial components and their derivatives. Interleukin (IL)-4 suppressed both m-TNF production and TNF-α secretion. p-Toluene- sulfonyl-L-arginine methyl ester (TAME) inhibited specifically TNF-α secretion and not m-TNF expression. Although blood monocytes produced small amounts of m- TNF, monocyte-derived macrophages showed enhanced m-TNF after cultivation with GM-CSF for 10 days. These findings indicate that m-TNF is expressed as a step in the TNF-α producing system, and suggest that m-TNF may play important roles in exhibition of macrophage function in situ. J. Leukoc. Biol 53: 29–36; 1993.