Tumor induction by murine sarcoma virus in AKR and C58 mice. Reduction of tumor regression associated with appearance of Gross leukemia virus pseudotypes.

Adult AKR and C58 mice injected intramuscularly with murine sarcoma virus, Moloney isolate (M-MSV), developed high incidence of nonregressing local tumors. Histologically, these tumors revealed the typical pleomorphism of M-MSV sarcomas; in some cases, however, neoplastic tissue showed a nodular or diffuse growth of monomorphic myoblastlike cells, reminiscent of clonal aggregates. No depression of immune reactivity was found in M-MSV-injected mice as evaluated by direct hemolytic plaque-forming cells against SRBC and by virus-neutralizing antibody production. The MSV recovered from the induced tumors proved to be, by neutralization assay, a Gross (G)-MSV pseudotype. Moreover, tumor cell suspensions absorbed out cytotoxic antibody directed against G-cell surface antigens. Therefore, the conclusion was drawn that MSV with envelope characteristics of endogenous G leukemia virus had formed in vivo through a phenotypic mixing phenomenon. The failure of tumors to regress has been interpreted as mainly due to the partial unresponsiveness of host immune reactivity towards G-MuLV specified antigens. Since MSV-tumors arose in AKR mice after a very long latent period, the possibility was considered that this relative resistance might depend on immunologic mechanisms. In fact, M-MSV-injected AKR mice immunodepressed by goat antimouse lymphocyte serum or rendered partially tolerant by neonatal M-MuLV inoculation developed sarcomas with higher incidence and with a shorter latency. Furthermore, the MSV recovered from these early tumors proved to be the original Moloney pseudotype.

Accelerated neutrophil apoptosis in mice lacking A1-a, a subtype of the bcl-2-related A1 gene.

To elucidate the role of A1, a new member of the Bcl-2 family of apoptosis regulators active in hematopoietic cell apoptosis, we established mice lacking A1-a, a subtype of the A1 gene in mice (A1-a-/- mice). Spontaneous apoptosis of peripheral blood neutrophils of A1-a-/- mice was enhanced compared with that of either wild-type mice or heterozygous mutants (A1-a+/- mice). Neutrophil apoptosis inhibition induced by lipopolysaccharide treatment in vitro or transendothelial migration in vivo observed in wild-type mice was abolished in both A1-a-/- and A1-a+/- animals. On the other hand, the extent of tumor necrosis factor alpha-induced acceleration of neutrophil apoptosis did not differ among A1-a-/-, A1-a+/-, and wild-type mice. The descending order of A1 mRNA expression was wild-type, A1-a+/-, and A1-a-/-. Taken together, these results suggest that A1 is involved in inhibition of certain types of neutrophil apoptosis.

Augmentation of specific cell-mediated immune responses to tumor cells in tumor-bearing rats pretreated wih the antileukemia drug busulfan.

Lethal growth of a syngeneic transplanted tumor (KMT-17) was inhibited in inbred WKA rats pretreated with the antileukemia drug busulfan (BU). However, the lethal growth of KMT-17 was not inhibited by pretreatment with cyclophosphamide, adriamycin, or ftorafur. With the Winn assay, spleen cells from BU-pretreated KMT-17-bearing rats (TBR) inhibited the growth of admixed KMT-17 cells more strongly than did spleen cells from BU-untreated TBR. The augmented tumor inhibitory activity of spleen cells was KMT-17-specific, and this activity was abrogated by in vitro treatment of spleen cells with anti-T serum and guinea pig complement. Augmentation of the immune response to KMT-17-associated antigen(s) in BU-pretreated TBR was also demonstrated in lymphocyte-mediated cytotoxicity, as detected by a 51Cr release assay and by a delayed-type hypersensitivity with a radioisotope footpad assay. Tumor regression in BU-pretreated rats was demonstrated to be mediated by the augmentation of T-cell-mediated immune responses to tumor-associated antigens. The tumor inhibitory effect of BU was abrogated by adoptive transfer with thymus cells from normal rats but not with those from BU-pretreated rats 1 day before tumor inoculation. The augmentation oif the antitumor immune responses by pretreatment with BU was suggested to be due to the fact that BU selectively inhibited the suppressor cells of their precursors.

Spontaneous regression of syngeneic transplanted tumors in rats pretreated with the antileukemia drug busulfan.

Lethal growth of a syngeneic transplanted tumor (KMT-17) was inhibited in WKA rats pretreated with the antileukemia drug busulfan. The effect of busulfan was short-lasting, and administration of the drug after tumor challenge had little effect on the lethal growth of the tumor. After spontaneous regression of the tumor in rats pretreated with busulfan, specific transplantation resistance to identical tumor was acquired. Busulfan did not exhibit its effect in rats irradiated before tumor challenge. As shown by the Winn test, spleen cells from rats immunized with inactivated tumor cells after busulfan treatment inhibited growth of admixed tumor cells more strongly than did spleen cells from rats immunized without busulfan treatment. Enhancement of immune response to tumor cells rather than tumoricidal activity by busulfan was suggested.

Activation of cytotoxic polymorphonuclear leukocytes by in vivo administration of a streptococcal preparation, OK-432.

Injection ip of a streptococcal preparation, OK-432, into WKA and DONRYU rats induced in vitro cytotoxicity of polymorphonuclear leukocytes (PMN) against tumor cells in terms of their cytostasis and cytolysis. PMN were obtained from peritoneal exudates of WKA and DONRYU rats. Cytotoxicity of activated PMN was immunologically nonspecific, although a certain target selectivity was observed in PMN cytotoxicity. The titer of cytotoxicity of PMN was dependent on the dose of injected OK-432 and on the number of PMN in the cytotoxicity assay. Activated PMN appeared very early (6 hr) after OK-432 injection, and the cytotoxic titer of PMN decreased from 24 hours and disappeared at 96 hours after injection of the reagent. In vitro culture of PMN with OK-432 also evoked cytotoxicity of PMN.

Regression of established tumors in rats by injection of diethylaminoethyl-dextran and Friend murine leukemia virus.

Subcutaneously established tumors in WKA rats were treated with polycation DEAE-dextran (DEAE-D) and Friend murine leukemia virus (F-MuLV). This idea was based on "xenogenization of tumors," which is defined as the immunologic regression of transplanted tumors in syngeneic rats after artificial infection of tumors with murine leukemia viruses. Regressions of subcutaneously established tumors were induced in 13 of 40 (33%) rats by injection of DEAE-D and F-MuLV. Intratumor injections of DEAE-D and F-MuLV increased the regression of tumors in 7 of 12 (58%) rats as compared to that of tumors in 6 of 28 (21%) rats treated with DEAE-D and F-MuLV by other injection routes. Electron-microscopic and immunofluorescence examinations revealed that tumor cells were infected with F-MuLV and acquired F-MuLV-related surface antigen on the cell surfaces. Therefore, the regression in rats of subcutaneously established tumors by the injection of DEAE-D and F-MuLV may have been due to an immunologic mechanism that may have been the same as the xenogenization of transplanted tumors previously infected with murine leukemia viruses.

Change of antigenic expression on rat tumor cells after their transplantation.

Antigenic expression of 3-methylcholanthrene-induced transplantable fibrosarcoma KMT-17 cells was investigated in relation to days after ip transplantation. Cytotoxicity tests with antiserum against tumor-associated surface antigen of KMT-17 cells revealed that cytotoxic sensitivity and absorbing capacity decreased after transplantation, but they increased when other normal rats were given transplants of tumor cells. A decrease in the sensitivity was observed when immunosuppressively irradiated rats were given tumor transplants. Tumor cell density in the abdominal cavities of rats directly and absorbing capacity of KMT-17 cells to antiserum against the histocompatibility antigen did not change after transplantation. The possible mechanisms of antigenic change of KMT-17 cells were discussed.

Natural occurrence of lymphocytes showing cytotoxic activity to BALB/c radiation-induced leukemia RL male 1 cells.

Spleen cells from untreated young male and female C57BL/6 and C58 mice and of male C3H/He mice showed cytotoxic activity in vitro against BALB/c X-radiation-induced leukemia RL male 1 cells by 51Cr-releasing lymphocyte-mediated cytoxicity (LMC) tests, but old mice of these strains lacked LMC activity. In contrast, spleen cells from male and female AKR, BALB/c, and DBA/2 mice, and from female C3H/He mice had no appreciable LMC activity. The proportion of active cells in spleens from young (C57BL/6 times BALB/c)F1 or reciprocal hybrid mice was higher in females than in males. The specificity of the LMC reaction of RL male 1 cells, determined by LMC inhibition assays, was somewhat different from that of previously reported serologic X.1 tests. Thus the antigen detected by LMC has been tentatively designated X.1'. The main effector cells in this system were uncharacterized cells not adherent to glass surfaces or nylon-wool columns. These findings in RL male 1 leukemia extend the evidence for the presence of naturally occurring LMC. With the single unexplained exception of strain C3H/He, the LMC activity against RL male 1 cells, exhibited by untreated mice of various strains, corresponded with a previous classification of mouse strains immunologically as X.1 responders or as X.1 nonresponders according to their ability to reject X.1-positive leukemia cells.

Modulation of the immune response to transplanted tumors in rats by selective depletion of neutrophils in vivo using a monoclonal antibody: abrogation of specific transplantation resistance to chemical carcinogen-induced syngeneic tumors by selective depletion of neutrophils in vivo.

The role of neutrophils in transplantation immunity to syngeneic rat tumors was examined using a monoclonal antibody (RP-3) that depletes rat neutrophils selectively in vivo. We used 2 chemical carcinogen-induced transplanted tumors of different antigenic specificity (KMT-17 and KDH-8 of WKA rat origin). When neutrophils were selectively depleted by i.p. injection of RP-3 at the time of in vivo priming with X-irradiated tumor cells, the growth of subsequently s.c. transplanted identical tumors was not inhibited, in contrast to the group of rats immunized without RP-3 treatment. Tumor growth was also not inhibited when the immune rats were treated with RP-3 at the time of identical viable tumor cell challenge. These results suggest that neutrophils play a role in both the priming and effector phases of specific transplantation resistance to syngeneic tumors.

Na+/H+ exchange modulates rat neutrophil mediated tumor cytotoxicity.

Rat neutrophils stimulated with phorbol 12-myristate 13-acetate, Bacillus Calmette-Guérin, zymosan A, and beta-1,3-D-glucan from Alcaligenes faecalis showed cytotoxicity to various tumor cells. Hydrogen peroxide was shown to be an effector molecule in tumor cytotoxicity by inhibition using various active oxygen scavengers. The following findings suggest that tumor cytotoxicity by rat neutrophils stimulated with the four reagents mentioned above is regulated by Na+/H+ exchange: (a) an increase in extracellular pH (pHo) from 6.5 to 8.0 resulted in enhancement of both tumor cytotoxicity and H2O2 production; (b) amiloride and its derivatives, inhibitors of Na+/H+ exchange, inhibited both functions of neutrophils mentioned above; (c) amiloride reduced intracellular pH (pHi) of neutrophils stimulated with the four reagents; (d) a decrease in the extracellular concentration of Na+ [( Na+]o) inhibited H2O2 production; (e) monensin, a Na+/H+ exchange ionophore, enhanced tumor cytotoxicity by neutrophils.

Tumor cytotoxicity of polymorphonuclear leukocytes in beige mice: linkage of high responsiveness to linear beta-1,3-D-glucan with the beige gene.

Beige mice (bg/bg) have many functional defects in their leukocytes and these phenotypes are inherited autosomal recessively. We studied the tumor cytotoxicity of polymorphonuclear leukocytes (PMN) obtained from bg/bg. The intensity of tumor cytotoxicity of PMN induced by linear beta-1,3-D-glucan was significantly higher in bg/bg PMN than in PMN of heterozygous control mice (bg/+). To analyze this phenomenon more precisely from the genetic viewpoint, we determined the tumor cytotoxicity of PMN from mice obtained by several mating experiments. (a) The intensity of linear beta-1,3-D-glucan-induced PMN cytotoxicity was found to be genetically defined and linked completely with the beige gene. In litter mates obtained from bg/+(female) X bg/bg(male), bg/bg(female) X bg/+(male), and bg/+(female) X bg/+(male) mating, PMN from only bg/bg showed significantly higher tumor cytotoxicity than those from bg/+ or mice that do not possess the beige gene (+/+). (b) The tumor cytotoxicity induced by other stimulants (phorbol myristate acetate and cytokines) was not significantly higher in bg/bg than bg/+ or +/+ PMN. It was concluded that the high responsiveness to linear beta-1,3-D-glucan in terms of tumor cytotoxicity of PMN was determined by the locus that is linked to the beige gene and is expressed autosomal recessively.

In vivo antitumor effect of lymphokine-activated rodent polymorphonuclear leukocytes.

In vivo tumor inhibitory activity of polymorphonuclear leukocytes (PMN) treated in vitro with lymphokine(s) (LK) was investigated with Winn's assay. Culture supernatants of BALB/c mouse spleen cells incubated with a streptococcal preparation, OK-432, were used as an LK source. With the use of a [3H]uridine release assay, RL male-1 tumor cells were lysed to some extent by peritoneal BALB/c mouse PMN treated with this LK preparation. With Winn's assay, LK-treated PMN from BALB/c mice completely inhibited the growth of the admixed syngeneic tumor at a high effector to target ratio, when normal mice were used as recipients. When X-irradiated mice or nude mice were used as recipients, the tumor growth was partially inhibited by admixed LK-treated PMN, but the tumor began to grow gradually and finally killed the recipient mice, even when a high effector target ratio was used. When nude mice which had been given i.v. transfers of nylon wool column effluent spleen cells were used as recipients, the tumor inhibitory activity of LK-treated PMN was recovered to the same level as when normal mice were used as recipients. On the other hand, tumor inhibition by admixed LK-treated PMN in nude mice was not recovered by the transfer of X-irradiated nylon column effluent T-cells. As a mechanism of tumor inhibition by LK-treated PMN, a possible role of LK-treated PMN in reduction of tumor load is discussed.

Production of factor(s) that render polymorphonuclear leukocytes cytostatic from spleen cells stimulated with a streptococcal preparation, OK-432.

We previously reported the augmentation of tumor cytotoxicity of polymorphonuclear leukocytes (PMN) by in vivo administration of a streptococcal preparation, OK-432 (S. Watabe et al., J. Natl. Cancer Inst., 72: 1365-1370, 1984). The present study was undertaken to elucidate the mechanisms of the phenomena. Mouse and rat spleen cells were stimulated in vitro with OK-432. The culture supernatants from the stimulated spleen cells (OK sup) contained factor(s) that rendered mouse and rat PMN cytostatic [neutrophil activating factor (NAF)]. The stimulation of spleen cells with a small dose of OK-432 (0.05 micrograms/ml) resulted in the production of maximum NAF, and NAF was produced soon (12 h) after OK-432 stimulation. NAF was partially inactivated with 60 degrees C 30-min treatment, and completely inactivated with 100 degrees C 10 min. NAF was sensitive to pH 2 treatment. The treatment of PMN with OK sup for 5 min at 37 degrees C was sufficient to induce cytostatic activity of PMN. That OK sup contained gamma-interferon and recombinant gamma-interferon showed NAF activity indicate that gamma-interferon is a NAF in OK sup.

Effect of combination treatment with cyclophosphamide and nonspecific passive immunization on a transplantable tumor in WKA rats.

Combination of cyclophosphamide (CY) and passive immunization with lymphoid cells sensitized to allogeneic tumor was studied in the treatment of a methylcholanthrene-induced transplantable fibrosarcoma in WKA rats. For determination of the most effective timing of combination treatment, rats given an injection of CY on Day 3 received passive transfer of the sensitized lymphoid cells on Day 0, 1, 2, 3, 4, or 6. A remarkable therapeutic effect was observed only when the passive transfer was combined on Day 4 with CY on Day 3. Rats inoculated with tumor succumbed in all cases without any treatment. After i.v. injection of CY on Day 3, 2 of 28 rats were cured (7.1%). Passive immunization with the sensitized lymphoid cells on Day 4 resulted in no therapeutic effect. After combination of CY on Day 3 and transfer of nonsensitized normal lymphoid cells on Day 4, 2 of 15 rats survived (13.3%). However, combination of CY and passive transfer of the nonspecifically sensitized lymphoid cells resulted in 23 survivors of 29 rats (79.3%).

Enhancement of antitumor transplantation resistance in rats by appropriately timed administration of busulfan.

Enhancement of specific transplantation resistance to a syngeneic tumor (KMT-17) was observed in WKA rats by treatment with the antileukemia drug busulfan (BU) (15 mg/kg) 5 days before and 5 days after immunization with X-irradiated KMT-17 tumor cells. Rats immunized with X-irradiated KMT-17 cells and then treated with BU showed specific transplantation resistance only against KMT-17 tumor. Carrageenan administration after BU treatment had no effect on enhancement by BU, which indicated that macrophages were not playing a major role in the observed enhancement. With the Winn assay, it was found that spleen cells from rats immunized with X-irradiated tumor cells followed by BU inhibited the growth of admixed tumor cells more strongly than did spleen cells from rats only immunized or only BU treated and that the tumor-neutralizing activity of spleen cells from rats treated by immunization followed by BU was abrogated by treatment with anti-T-serum and complement. It was suggested that the enhanced antitumor transplantation resistance caused by BU was due to enhanced T-cell immune responses to tumor cells. Enhancement of anti-tumor transplantation resistance by BU was significantly abrogated by adoptive transfer with thymus cells and was slightly abrogated with spleen cells from rats immunized with X-irradiated KMT-17 cells 1 day before tumor challenge but receiving no other treatment. Transfer of sera from the immunized rats had no effect on enhancement by BU. These results, taken together, suggest that the mechanism of the enhancement by BU involved a selective elimination of the immunosuppressor cells from the immunized hosts.

Regulation of leukocyte adherence and migration by glycosylphosphatidyl-inositol-anchored proteins.

Leukocyte extravasation is essential for subsequent inflammation and the immune response. Extravasation can be divided into at least three steps; rolling, firm adhesion, and transendothelial migration. Although the mechanisms involved in the first two steps have been fairly well documented, the last step is complex and largely remains to be clarified. This review focuses on the possible role of GPI-anchored proteins on leukocytes in the regulation of their transendothelial migration. In addition to regulation by urokinase and its receptor, which has been regarded as the main modulator, we draw attention to a novel GPI-anchored protein (GPI-80) on human phagocytes that may be involved in regulating leukocyte adhesion and migration. The high degree of homology of GPI-80 with vanin-1, which is expressed on vascular tissues and is involved in prethymic cell homing into the thymus, raises the possibility that there is a family of molecules, including GPI-80 and vanin-1, that may be involved in leukocyte transendothelial migration. The possible role of soluble GPI-anchored proteins in this process is also discussed.

Suppression of late phase enhanced vascular permeability in rats by selective depletion of neutrophils with a monoclonal antibody.

We investigated the role of neutrophils in increased vascular permeability by a selective reduction of neutrophils using a monoclonal antibody, RP-3. An intraperitoneal injection of RP-3 not only selectively depleted peripheral blood neutrophils, but prevented the neutrophil infiltration to the tissues. Proteose peptone, zymosan, and BCG induced three different types of inflammatory edema, showing the early phase only, early plus late phase, and the late phase only, respectively. Only the late phase response of zymosan and BCG was inhibited by a depletion of neutrophils by RP-3, though the early phase response induced by proteose peptone and zymosan was not affected. Reconstitution of neutrophil-depleted rats by in situ injection of these cells restored the inflammatory edema induced by BCG, depending upon the number of neutrophils injected.

Neutrophil secretory vesicles are the intracellular reservoir for GPI-80, a protein with adhesion-regulating potential.

The subcellular localization of GPI-80, a novel, adhesion-regulating protein, was investigated in human neutrophils. Surface expression of GPI-80 was determined by FACS analysis as well as by the ability for phospholipase C to cleave the protein from the cell surface. Increasing amounts of GPI-80 were exposed on the cell surface after weak stimulation with the chemoattractant fMLF, suggesting that the protein can be translocated to the plasma membrane from intracellular stores. By subcellular fractionation of the neutrophils, GPI-80 was defined as a component of a light membrane fraction, containing secretory vesicles and plasma membranes, and it was absent from the neutrophil granule fractions. Separation of the plasma membranes from the secretory vesicles by flotation gradient fractionation confirmed that the GPI-80 was localized in the mobilizable secretory vesicles by approximately 50%, and the rest was plasma membrane-bound. Thus, we identify secretory vesicles as the reservoir of GPI-80 from which it may translocate to the plasma membrane after weak stimulation of the cells.

Modulation of rat neutrophil functions by administration of granulocyte colony-stimulating factor.

Neutrophil function was examined in rats treated with recombinant human granulocyte colony-stimulating factor (G-CSF) using peripheral blood neutrophils (PBNs) and peritoneal exudate neutrophils (PENs) as sources of cells examined in vitro. Adherence to plastic plates containing fetal calf serum of nonstimulated or N-formylmethionyl-leucyl-phenylalanine (fMLP)- or tumor necrosis factor-alpha-stimulated PBNs obtained from G-CSF-injected rats was lower than that of control rats. In contrast, this adherence was higher in G-CSF-treated rats than in the control group when PENs were used. Neutrophil adherence of G-CSF-injected and noninjected groups was identical when phorbol myristate acetate was used to stimulate neutrophils. Superoxide production of PBNs stimulated with fMLP in vitro was lower in G-CSF-treated rats than in control rats but higher than in the controls when PENs were used. Furthermore, in vitro tumor cell growth inhibition by PBNs was lower in G-CSF-treated rats than in control rats, but when PENs were used inhibition was higher than in the controls.

Clustering on the forward surfaces of migrating neutrophils of a novel GPI-anchored protein that may regulate neutrophil adherence and migration.

We previously reported a novel glycosylphosphatidylinositol (GPI)-anchored glycoprotein (tentatively designated GPI-80) on human leukocytes that may be involved in the regulation of neutrophil adherence and migration. In this study, we examined by immuno- and scanning electron microscopy, the distribution of GPI-80 on neutrophil surfaces. GPI-80 was diffusely distributed on the surface of resting neutrophils and on the peripheral areas of adherent cells after stimulation with N-formyl-methionyl-leucyl-phenylalanine. After longer stimulation (60 min), GPI-80 decreased in number and was again diffusely distributed on the surfaces of round neutrophils. Few GPI-80 were detected on the ventral surfaces of adherent neutrophils. Clusters of GPI-80 were detected on the forward surfaces of neutrophils transmigrating through pores of nitrocellulose membranes. These results may give a morphological background of possible role of GPI-80 for neutrophil extravasation.