Akio Okubo

Heterogeneity in responses of human blood monocytes to granulocyte-macrophage colony-stimulating factor.

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.

Production of Tumor Necrosis Factor-α by Alveolar Macrophages of Lung Cancer Patients

The abilities of human alveolar macrophages (AM) obtained from healthy donors and patients with lung cancer to produce tumor necrosis factor (TNF) were compared with those of their blood monocytes after activation with lipopolysaccharide (LPS). TNF activity was assayed by measuring cytotoxicity against actinomycin D‐treated L929 cells and TNF was determined quantitatively by sandwich enzyme‐linked immnnosorbent assay (ELISA) with polyclonal and monoclonal antibodies against TNF‐α. Unstimulated AM from healthy donors released variable amounts of TNF spontaneously, whereas blood monocytes did not. When treated with LPS for 24 h, AM and monocytes produced TNF dose‐dependently, but TNF production by AM was significantly more than that by blood monocytes. This TNF activity was inhibited completely by monoclonal anti‐TNF‐α antibody. Macrophages generated by in vitro maturation of monocytes induced by granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) produced more TNF than freshly isolated monocytes. No difference was found in the abilities of AM from healthy donors and patients with lung cancer to produce TNF after activation stimuli. These observations suggest that human AM may be important in in vivo antitumor defense of the lung through TNF‐α production.

Tumor cytotoxicity and interleukin 1 production of blood monocytes of lung cancer patients

SummaryThe effects of lung cancer on the abilities of blood monocytes to produce interleukin-1 and to mediate antitumor activity were examined. The functional integrity of blood monocytes was determined by their capacity to respond in vitro to a variety of activating agents and become tumoricidal, as assessed by a radioactive release assay and ability to produce interleukin-1 in vitro. The results show that the presence of lung cancer significantly increased the number of harvested blood monocytes and that the spontaneous tumoricidal activity of these monocytes was slightly high as compared to monocytes obtained from healthy donors. The production of interleukin-1 by monocytes of healthy donors and lung cancer patients was similar. Blood monocytes obtained from lung cancer patients were less cytotoxic against allogeneic A375 melanoma cells as compared with those of healthy donors subsequent to incubation with a soluble muramyl dipeptide analog or lipopolysaccharide, but were as tumoricidal as those from healthy donors when activated with lipophilic muramyl tripeptide (MTP-PE) entrapped in multilamellar liposomes. The finding that monocytes of patients with lung cancer can respond to MTP-PE encapsulated in liposomes, recommends the use of these liposomes in therapy of human lung cancer.

Killing of Alveolar Macrophages and of Monocytes that Have Responded to Granulocyte‐Macrophage Colony‐stimulating Factor by Human Lymphokine‐activated Killer Cells

The susceptibilities of human blood monocytes and alveolar macrophages (AM) to cytotoxicity mediated by lymphokine (IL‐l)‐activated killer (LAK) cells were examined. Monocytes and AM of healthy donors were obtained by counter‐flow centrifugal elutriation (CCE) and bronclioalvcolar lavage, respectively. The LAK activity induced by incubation of blood mononuclear cells (MNC) for 4 days with recombinant interleukin 2 (IL‐2) was measured by a 4‐h 51Cr release assay. The LAK cells were not cytotoxic to freshly isolated monocytes, but were cytotoxic to autologous fresh AM and monocytes that had been incubated for more than 4 days in medium alone. Blood monocytes that had been incubated for 4‐ days in medium with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), macrophage colony‐stimulating factor (M‐CSF) or interleukin 3 (IL‐3) were much more susceptible than untreated monocytes to the cytotoxicity of LAK cells. When blood monocytes were separated by CCE into subpopulations of three sizes (small, medium and large), the medium‐ and large‐sized monocytes showed greater responses to GM‐CSF in terms of DNA synthesis and colony formation than the small‐sized cells. After treatment with GM‐CSF for 4 days, these medium and large monocytes were more susceptible than the small monocytes to the cytotoxic action of LAK cells. These results suggest that LAK cells may be important in situ in down‐regulating the functions of mature macrophages and blood monocytes that have responded to GM‐CSF.

Tumor cytotoxicity of human monocyte membrane-bound interleukin-1α induced by synergistic actions of interferon-γ and synthetic acyltripeptide, FK-565

SummaryHuman blood monocytes were isolated by counter-flow centrifugal elutriation from healthy donors and these noncytotoxic monocytes were rendered tumoricidal to allogeneic melanoma (A375) cells by activation with a synthetic acyltripeptide (FK-565), as assessed by measuring release of [125I]iododeoxyuridine in 72 h. When monocytes were treated with FK-565 for 16 h, and then fixed with paraformaldehyde, they showed cytotoxicity to A375 melanoma cells. The fixed-monocyte-mediated cytotoxicity to A375 cells was induced by the synergistic actions of FK-565 and recombinant interferon-γ (rIFN-γ), but not other cytokines [rIFN-αA, rIFN-β, tumor necrosis factor (TNF), interleukin (IL)-2, -3 and -6]. For synergistic activation of monocytes with induction of a membrane-associated antitumor monokine, the monocytes had to be incubated first with rIFN-γ and then with FK-565. FK-565 also acted synergistically with rIFN-γ to stimulate monocytes to produce membrane-associated IL-1 activity, which induced C3H/HeJ thymocyte blastogenesis in response to phytohemagglutinin P. The tumoricidal and thymocytestimulating activities of the fixed monocytes were almost completely inhibited by a specific anti-(IL-1α) antiserum, but not by a specific anti-(IL-1β) antiserum or monoclonal anti-TNF antibody. These results suggest that membrane-associated IL-1α of human blood monocytes can be induced by two activation signals (rIFN-γ then FK-565) at their suboptimal concentrations.

Synergism of synthetic acyltripeptide and its analogs with recombinant interferon γ for activation of antitumor properties of human blood monocytes

SummaryHuman blood monocytes freshly isolated by centrifugal elutriation from healthy volunteers were not cytotoxic to allogeneic A375 melanoma cells, but they were activated to the tumoricidal state by incubation in vitro with FK-565, (heptanoyl-γ-D-Glu-(L)-meso-α,ε-A2pm(L)-D-AlaOH), which is a synthetic acyltripeptide closely resembling cell wall peptidoglycan peptides of Streptomyces in structure. Among 11 different derivatives of FK-565, 7 analogs were more potent activators of monocytes for tumor cell killing than FK-565. The maximal expression of tumoricidal nonocytes was dependent on the concentration of FK-565 or its analogs added and the ratio of monocytes to target tumor cells. In a parallel experiment, a combination of a subthreshold concentration of FK-565 or its analogs (FR-42148 and FR-42149) and recombinant interferon γ (rIFN-γ) induced significant monocyte-mediated tumorcell killing, indicating that the effects of rIFN-γ and acyltripeptide or its analogs in monocyte activation are synergistic. In contrast to rIFN-γ, recombinant rIFN-αA and rIFN-β had additive effects with acyltripeptide or its analogs in human monocyte activation. These results suggested that synthetic acyltripeptide and its analogs combined with rIFN-γ could be of clinical value for in situ activation of the tumoricidal activity of human blood monocytes responsible for eradication of cancer metastases.