Yoshimi Shibata

Differential induction of prostaglandin E2-dependent and -independent immune suppressor cells by tumor-derived GM-CSF and M-CSF.

Both prostaglandin E2 (PGE2)‐dependent (indomethacin‐sensitive) and PGE2‐independent (indo‐ methacin‐insensitive) suppressor cell activities that inhibited mitogenic T cell blastogenesis appeared in the bone marrow and spleen of mice on days 20 to 30 following transplantation of NFSA fibrosarcoma molecularly expressing mRNA for both macrophage (M) and granulocyte‐macrophage (GM) colony‐stimulating factors (CSFs). The present study was done to characterize the two different suppressor cells isolated from NFSA tumor‐bearing mice and to verify a role of CSFs in the induction of suppressor cells in vitro. Whereas PGE2‐releasing suppressor cells were found in bone marrow and spleen cells isolated from tumor‐bearing mice, indomethacin‐insensitive suppressor cells in both tissues were localized predominantly in adherent cell fractions. An increase in Mac‐1+ and Mac‐2+ spleen cell populations with two to three times larger cell volumes was observed, and both showed strong PGE2‐releasing capacity and indomethacin‐sensitive suppressor cell activity. However, after elimination of Mac‐1+ or Mac‐2+ cells, bone marrow cells still showed higher PGE2‐releasing capacity and indomethacin‐sensitive suppressor activity. The in vitro cultures of normal bone marrow and spleen cells with NFSA cell conditioned medium (NFSA‐CM) induced heterogeneous mixtures of indomethacin‐sensitive and ‐ insensitive suppressor cells like those observed in cultures with the combination of M‐CSF and GM‐CSF. However, cultures with either GM‐CSF or M‐CSF resulted in the induction of indomethacin‐sensitive suppressor cells by GM‐CSF and of indomethacin‐insensitive suppressor cells by M‐CSF. In addition, NFSA‐CM pretreated with anti‐GM‐CSF antibody induced indomethacin‐ insensitive suppressor cells in in vitro cultures of bone marrow and spleen cells. These results suggest that two distinctly different suppressor cells developed under hemopoiesis of myelomonocytic lineage cells are regulated differentially by the two macrophage growth factors, M‐CSF and GM‐CSF

Selectively eliminated blood monocytes and splenic suppressor macrophages in mice depleted of bone marrow by strontium 89

The contribution of specific activity to the effects of the bone‐seeking isotope, strontium 89 on radiosensitive components of mononuclear phagocyte populations was investigated in mice. CBA/J mice received a fixed dose of 2 μCi/g body weight of 89Sr with three different specific activities, 6 Ci, 100 μCi and 20 μCi per mg Sr. The estimated radioactivity located in the bone surface was 4,200, 3,000 and 2,400 cpm/mg bone when measured 2 days after the administration of 89Sr, and was lost with an estimated biological half‐life of 27, 25, and 23 days, respectively. Bone marrow suppression was assessed by quantitation of the depletion of macrophage‐colony forming cells (M‐CFC) grown in vitro in the presence of macrophage growth factor. The decline in M‐CFC closely paralleled the level of radioactivity in the bone. These effects were clearly reflected by the depletion of monocytes in the blood, which were reduced to 14%, 14%, and 21% of control levels corresponding to SAs of 6 Ci/mg, 100 μCi/mg and 20 μCi/mg when counted on day 10. By day 30 the respective monocyte levels were 15%, 31%, and 77%. Furthermore, the induction of prostaglandin E producing suppressor macrophages (Mø) by Corynebacterium parvum administration was found to vary inversely with the effects of radioactivity in the bone, with initial impairment followed by quantitative recovery. Resident‐type Mø in peritoneal cavity, however, appear to be unaffected by 89Sr‐treatment. These data suggest, as before, that the monocytes and suppressor Mø are dependent on radiosensitive marrow cells. The observations also lead to the conclusion that the specific activity of 89Sr preparations is an important determinant of the degree of suppression and of the rate of recovery of bone marrow from the effects of irradiation that follow the administration of this isotope.

Relationships between suppressor macrophages and macrophage precursors in the spleens from tumor-bearing mice

Suppressor macrophages (M phi) which can inhibit mitogen-induced lymphocyte proliferation appeared in the spleens of mice bearing transplanted MC-A fibrosarcoma cells. An analysis of the ontogeny of such M phi revealed additional suppressor activity directed against macrophage stem cells. Treatment of spleen cell suspensions with carbonyl iron followed by centrifugation removed suppressor M phi but did not deplete M phi-colony forming cells (M-CFC) which could be demonstrated in soft agar culture in L-cell conditioned medium (LCM). Untreated spleen cells had normal numbers of M-CFC; phagocyte-depleted mononuclear cells showed a threefold increase in M-CFC 14 days after subcutaneous inoculation of 10(6) MC-A cells per mouse. Further increases in M-CFC were also evident in similar preparations on Days 21 and 28 when the M-CFC concentration reached a maximum of eight times the normal level. The M phi which developed from the M-CFC grown in the presence of LCM were later shown to have indomethacin-sensitive suppressor activity suggesting the mediation of this phenomenon by prostaglandins. These observations suggest that locally produced phagocytic suppressor M phi from the spleens of tumor-bearing mice play important roles not only as inhibitors of lymphocyte proliferation as reported earlier, but also as regulators of monocyte-M phi production.

Regulation of phospholipase A2 activation and arachidonic acid metabolism in an interleukin‐3–dependent macrophage‐like cell line

An interleukin 3 (IL‐3)–dependent macrophage‐like cell line, 11‐1‐B3, was newly established from CBA/J mouse bone marrow cell cultures. Assay of eicosanoids in the culture supernatants of the intact and [3H]arachidonic acid (AA)–prelabeled cells showed that, after stimulation with the Ca2+ ionophore A23187, the 11‐1‐B3 cells synthesized and released relatively large amounts of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) but not LTC4. In addition, 11‐1‐B3 cells showed Ca2+‐dependent and alkaline pH–optimal phospholipase A2 (PLA2) activity that preferentially hydrolyzed cleavage of sn‐2‐arachidonyl‐ but not sn‐2‐oleoylphosphatidylcholine. The cellular enzyme was distributed with 90% of the activity in the cytosol and 10% in the membrane fraction. Treatment of cells with A23187 for 5–10 min resulted in five‐ to sevenfold increases in the membrane‐associated PLA2 but activity in the cytosol was unchanged. This increase in membrane‐associated enzyme activity was transient, returning to the pretreatment distribution after 30 min. In sharp contrast, phorbol myristate acetate (PMA) stimulation failed to induce either eicosanoid release or PLA2 activation, although PMA induced translocation of protein kinase C (PKC) to the membrane fraction within 10 min. The data suggest that increases in cellular Ca2+ directly activate membrane‐associated PLA2 and consequently initiate AA metabolism; PKC activation by PMA requires additional steps to activate PLA2, a mechanism that is apparently deficient in the IL‐3–dependent Mø‐like cells.

Restoration of Prostaglandin Releasing Macrophage Populations in Lethally Irradiated Mice With Spleen Cells From Bone Marrow-Depleted Donors

Previous studies in mice severely depleted of bone marrow cells by 89Sr showed persistent monocytopenia and impaired expression of prostaglandin E2‐releasing splenic macrophages (PGSM) despite the occurrence in the spleen of more than 10‐fold increases in pluripotential stem cells and Mo colony‐forming cells. To determine whether the observed deficits were due to a lack of precursors of blood monocytes and PGSM in the spleens of 89Sr‐treated mice, radiation chimeras were established by iv infusion of 2 × 106spleen cells from 89Sr donor CBA/J or semisyngeneic B6CB F1 hybrid mice into lethally γ‐irradiated CBA/J recipients. Blood monocyte levels were greater than normal by day 14 and PGSM induced by Corynebacterium parvum were demonstrated by day 28. These restored Mø populations expressed the donor haplotype detected in vitro with haplotypespecific monoclonal anti‐H‐2K plus complement. “Sr treatment of the chimeras resulted in profound depletion of monocytes and PGSM. The data indicate that the spleen of the 89Sr‐treated mouse, which is an ineffective source of circulating monocytes and PGSM, contains cells which can generate both of these populations following infusion into lethally irradiated recipients. Since the bone marrow of such recipients was capable of being repopulated, the aggregate observations suggest that functional bone marrow is obligatory for the generation of blood monocytes and PGSM populations.

Effects of bone marrow ablation on compartmental prostaglandin synthesis by mononuclear phagocytes.

Mononuclear phagocyte functions were studied in mice selectively deprived of bone marrow and rendered profoundly monocytopenic by the administration of the bone seeking isotope, 89Sr. Characteristics of such mice include severe impairment of monocyte-M phi elicitation, ablation of C. parvum induction of PGSM but the persistence of resident peritoneal and pulmonary alveolar M phi populations; splenic M phi increase in number concomitantly with splenic hemopoiesis. Studies on compartmental regulation in this model suggest that the capacity of splenic M phi to synthesize and release PGE2 is dependent upon a function of the bone marrow and is not wholly determined by the local environment. The relationship of blood monocytes to PGSM is uncertain. In contrast to splenic M phi, the capacity of resident peritoneal M phi for eicosanoid synthesis appears to be independent of bone marrow function. Monocyte influx, moreover, does not appear necessary for the maintenance of the resident peritoneal and alveolar M phi populations. We do not yet know whether bone marrow ablation destroys a migratory precursor of PGSM or the source of a crucial regulatory agent. In conclusion, the observations discussed show that prostaglandin metabolism within the spleen is subject to extracompartmental influence. It is clearly important to determine the regulatory characteristics of individual M phi compartments and generalizations about functional properties of mononuclear phagocytes should be made with circumspection.