Alan M. Kaplan

Macrophage regulation of mitogen-induced blastogenesis: I. Demonstration of inhibitory cells in the spleens and peritoneal exudates of mice

Mitogen-induced blastogenesis of mouse B- and T-lymphocytes was inhibited by adherent spleen cells from normal or immunomodulator-treated mice. Blastogenesis could be similarly inhibited by the addition of syngeneic peritoneal exudate cells (PEC) from either normal or immunomodulator-treated mice suggesting a regulatory role for macrophages in lymphoproliferation. An adherent subpopulation of PEC (macrophages) was demonstrated to mediate inhibition of blastogenesis. Adherent PEC cells from immunomodulator (pyran copolymer)-treated mice had greater inhibitory activity than comparable cells from normal mice. Moreover, T-lymphocyte blastogenesis was more resistant to macrophage regulation than B-lymphocyte blastogenesis. Significant inhibition of blastogenesis could be demonstrated when ϑ -bearing cells were removed prior to the addition of PEC to cultures of spleen cells. Addition of nonadherent PEC to cultures of spleen cells was demonstrated to result in enhancement rather than inhibition of blastogenesis. Macrophage-mediated inhibition of blastogenesis was not merely the result of the potentiation of the effect of suppressive splenic macrophages since PEC were found to inhibit the response of adherence-depleted spleen cells even more than that of whole spleen cells.

Immunotherapy with autologous white cell infusions (“lymphocytes”) in the treatment of recurrent glioblastoma multiforme. A preliminary report

Autologous leukocytes (107 to 109), obtained with the Haemoneticss Leukaphoresis apparatus, were inoculated directly into recurrent glioblastoma tumors via indwelling catheters or by direct intratumoral injection through existing craniotomy openings. The rational use for autologous leukocyte (lymphocyte) infusions was based on in vitro autologous lymphocyte cytotoxicity to glioblastoma cells in the absence of serum inhibitory factors. Seven of 17 patients treated had life expectancy under 1 month; all patients had received definitive surgery, and all but two received radiation, nitrosourea chemotherapy and/or dexamethasone, and showed evidence of clinically recurrent disease. Following autologous leukocyte infusion (lymphocyte/granulocyte ratio 1:1), eight patients sustained clinical improvement and were alive up to 17 months later. No neurotoxicity ascribable to the procedure has been observed. One patient, who was comatose at the time of single leukocyte infusion, returned to full activity and lived for 17 months without an increase in tumor mass by brain scan. These results suggest that infusions of autologous leukocytes (lymphocyte‐monocytes) directly into glioblastoma may be a viable additional treatment for glioblastoma and certainly warrants further evaluation.

Macrophage regulation of mitogen-induced blastogenesis: II. Mechanism of inhibition

The addition of normal or immunomodulator-activated peritoneal macrophages has been shown to inhibit the in vitro mitogen-induced blastogenic responses of both T-and B-lymphocytes. Serial determinations of the number of viable cells per culture indicated that neither activated nor normal peritoneal macrophages were cytotoxic for lymphocytes in culture. The presence of blast cells could not be demonstrated in cultures incubated with macrophages, suggesting that macrophages acted by inhibiting blast formation rather than by selectively killing blast cells. Supernatant materials from 24-hr peritoneal exudate cell cultures had relatively little effect on blastogenesis compared to a comparable number of viable cells. However, supernatant materials from cocultures of peritoneal exudate cells and normal spleen cells significantly inhibited blastogenesis in LPS-stimulated cultures but not in PHA-stimulated cultures. Inhibition of blastogenesis by macrophages was an early event and inhibition could be demonstrated when macrophages were present for only the first 24 hr of culture. Pretreatment of macrophages with enzymes prior to addition to cultures of spleen cells resulted in a decrease of inhibition of blastogenesis suggesting that some surface molecule on the macrophage was required for inhibition.

Immunoadjuvant activity of pyran copolymer. I. Evidence for direct stimulation of t-lymphocytes and macrophages.

Abstract A single injection of pyran copolymer has been shown to greatly increase the number of hemolytic plaque forming cells to sheep erythrocytes (sRBC). Pyran given from 1 day before to 2 days after sRBC inoculation increased both specific activity and plaques/spleen, suggesting that macrophage activation was probably not responsible for the enhancement seen. In addition, pyran given 1 day prior to the primary injection of sRBC was found to increase the secondary response to SRBC given alone. As similar experiments using thymectomized irradiated bone marrow reconstituted mice showed no increase in specific activity following pyran administration, it was unlikely that pyran was acting directly on B cells. Furthermore, experiments measuring the antibody response to Escherichia coli lipopolysaccharide, a thymic independent antigen, pyran did not increase the response to this antigen. In contrast to the above, pyran delayed and depressed cell mediated cytotoxicity to the allogeneic DBA/2 P815 mastocytoma. However, no difference in the titers of cytotoxic antibody against mastocytoma cells was seen between pyran-treated and normal animals. Pyran was mitogenic for spleen cells in vitro . However, following the administration of pyran in vivo , mitogen induced blastogenesis in vitro to PHA and LPS was inhibited and this inhibition was determined to be macrophage-dependent. These results are consistent with a model in which the immunoregulatory effects of pyran act through macrophages and T-lymphocytes.

Effect of Macrophages on Fibroblast DNA Synthesis and Proliferation

Abstract Mouse peritoneal macrophages incubated with mouse 3T3 fibroblasts modulate DNA synthesis by the fibroblasts as measured by 125IUDR incorporation into DNA. The factor responsible for this activity is soluble and inhibits fibroblast DNA synthesis when present in high concentrations, whereas stimulation was observed at lower concentrations. Similar findings were observed when soluble fractions from human peripheral monocytes were incubated with primary human skin fibroblasts. In addition to increased DNA synthesis, there was a significant increase in human fibroblast proliferation as measured by direct cell counting. These findings support the hypothesis that macrophages play a regulatory role in wound healing through their capacity for positive and negative regulation of fibroblast DNA synthesis and proliferation.

MACROPHAGE MEDIATED TUMOR CELL CYTOTOXICITY

The following evidence from our research has implicated the macrophage as an important effector cell in pyran and/or C. parvum induced host resistance to solid tumors: (1) Increased infiltration of tumors with histiocytes following systemic treatment with pyran;17 (2) activated peritoneal macrophages with tumoricidal activity have been recovered from the peritoneal cavity of normal or tumor bearing mice treated with pyran or C. parvum;17 (3) activated peritoneal macrophages mixed with tumor cells in vitro and transplanted into syngeneic recipients inhibited tumor growth; (4) trypan blue, an inhibitor of macrophage function, prevent C. parvum induced regression of methylcholanthrene tumors; and (5) direct intralesional injection of activated macrophages into the MCA 2182 tumor inhibited tumor growth and increased the MST.

Human mononuclear phagocyte-associated antigens: II. Lymphokine-inducible antigens on the macrophage cell line, U937☆

Abstract We have utilized the U937 macrophage cell line as a model system for analysis of human mononuclear phagocyte (MNP) differentiation. In addition to expressing membrane antigens shared with other MNP, U937 possesses an intrinsic ability to become “activated” upon exposure to lymphokines. A heteroantiserum produced against lymphokine-stimulated U937 (anti-U937L) was utilized to detect acquired or inducible membrane antigens expressed on “activated” U937. Absorption of this antiserum to remove antibodies to nonstimulated U937 (U937N) did not remove the reactivity of anti-U937L/U937N to lymphokine-stimulated U937 as determined by an 125 I-protein A radioimmunoassay. The lymphokine-inducible antigens were not detectable on resident, human peritoneal macrophages. In addition to expression of lymphokine-inducible antigens, treated U937 cells displayed alterations in both morphology and functional activity (antibody-dependent cellular cytotoxicity). Kinetic analysis of lymphokine-stimulated U937 indicated that antigen expression occurred as early as 1–2 hr after lymphokine exposure, plateauing at 16–18 hr of stimulation. The inducible antigens were susceptible to proteolytic degradation and expression was blocked by inhibitors of protein synthesis. Inducible antigens detectable by anti-U937L/U937N did not result from the expression of cryptic or buried membrane antigens. Thus, the U937 cell line can be utilized for production of antibodies useful in analysis of membrane antigen expression during differentiation within the MNP system.

Human mononuclear phagocyte-associated antigens: I. Identification and characterization☆

Abstract Rabbit antisera were produced against a lymphokine-activated human macrophage cell line, U937 (αU937), and human peritoneal macrophages (αPEMO). After absorption with AB erythrocytes, pooled platelets, and B-lymphoblastoid cell lines, both antisera reacted by microcytotoxicity, indirect immunofluorescence (IF), and radioimmunoassay (RIA) with adherence-purified human peripheral blood monocytes, splenic and peritoneal macrophages, and leukemic myelomonoblasts. A panel of normal human T lymphocytes, B lymphocytes, and erythroid-myeloid or lymphoblastoid cell lines failed to react with both αU937 and αPEMO. Although both heteroantisera reacted against polymorphonuclear leukocytes (PMNs), after absorption with PMNs specific reactivity against mononuclear phagocytes remained. Absorption of αU937 and αPEMO with myelomonoblastic leukemia cells (AMML) removed IF and RIA activity against both PMNs and monocytes but not against splenic and peritoneal macrophages. In contrast, absorptions of both heteroantisera preparations with splenic macrophages abolished their IF and RIA reactivity not only to splenic and peritoneal macrophages but also to peripheral blood monocytes and leukemic myelomonoblasts. These results are consistent with (1) both antisera defining specific monocyte/macrophage-associated antigens(s) which are distinct from MHC-coded HLA-A,B,C, and DR antigens, and (2) expression of common monocyte/macrophage-associated antigen(s) and uniquely associated antigen(s) selectively expressed on tissue macrophages. These reagents will be useful in delineating human monocyte/macrophage differentiation as well as the immunological functions of mononuclear phagocytes.

SUPPRESSION OF THE HGG RESPONSE IN NORMAL MICE WITH A SPLEEN CELL LYSATE FROM TOLERANT MICE

Publisher Summary This chapter presents an analysis of the suppression of the human gamma globulin (HGG) response in normal mice with a spleen cell lysate from tolerant mice. In a study described in the chapter, tolerance to HGG was induced in A/J male mice by the intraperitoneal injection of 2.5 mg of ultracentrifuged DEAE-purified HGG. Spleen cells from tolerant or normal donors were either injected intraperitoneally into normal mice or subjected to sonication for 2 min on ice. The sonicate was centrifuged at 40,000g for 45 min, and the supernatants injected intravenously into normal recipients as the equivalent of 5 × 107 cells/ml. The response to HGG was determined after innoculation with heat aggregated HGG (AHGG) by the enumeration of antibody forming cells in a liquid hemolytic plaque assay. It was found that transfer of day 35 spleen cells or spleen cell lysates from tolerant donors to normal recipients resulted in the marked antigen specific suppression of the response to HGG. The response to HGG of recipients of tolerant spleen cell lysate was similarly suppressed.

Effects of Three Feeding Schedules on Tumor and Host of Lewis Lung Carcinoma-Bearing Mice

Abstract The effect of three feeding schedules on tumor and host were examined in Lewis Lung bearing (TB) and nontumor bearing (NTB) C57/Bl mice. Both NTB and TB animals were divided into three groups: the control groups which were fed ad libitum; the intermittent fed (IMF) groups were fed for 32 hr and fasted for 16 hr in each 48-hr cycle, and the alternate day fed (ADF) groups were fed for a 24-hr interval in each 48-hr cycle. The animals were killed at the end of the fifteenth day, following a fed day for all groups. In the NTB groups, only the ADF group showed decreased food intake and lower body weight gain as compared to their control group. In the TB mice, as compared to their control group, the IMF group showed a significant reduction in the mean tumor weight with no change in the mean host weight, even though the daily food intakes of these two groups were the same over the experimental interval. In contrast, the ADF group showed reductions in both host and tumor weights as compared to their control group. The tumor to host weight ratios were significantly reduced for both the IMF and ADF groups as compared to the ratios found for the control groups, which suggests a differential effect on the tumor and on the host due to the feeding schedule. As assessed by the protein, RNA, and DNA concentrations, no compositional differences were noted for the tumors obtained from the animals that were maintained on each of the three different feeding schedules. In the NTB mice, no differences in tissue leucine (Leu) oxidation occurred between the groups for liver and skeletal muscle, whereas in the TB animals in vitro Leu oxidation capability by skeletal muscle specimens was markedly enhanced in the ADF group, but no difference was noted for the IMF group of the TB mice when compared to the control group.