Carleton C. Stewart

Rodent peritoneal macrophages as bone resorbing cells

SummaryBecause of the difficulty in obtaining large, relatively pure populations of osteoclasts, most studies of bone resorption are performed on intact animals or in cultures of embryonic bone rudiments. These experimental systems, however, do not permit detailed analysis of the cellular mechanisms of matrix degradation or of the means whereby resorbing cells attach to the bone surface.Mononuclear phagocytes, which are probably ontogenetically related to the osteoclast, will resorb bone matrix in tissue culture. Consequently, we have developed an in vitro system whereby the ability of these cells to bind and resorb skeletal matrix can be precisely and individually measured using radioisotopically labeled, devitalized rat bone particles. We have found that when derived from mice, peritoneal macrophages bind approximately 80% of bone particles within the first 40 min of incubation. Significant (P<0.025) net matrix degradation, as defined by the percentage of isotope released from bone cultured with macrophages as compared to that released in the absence of cells, occurs within the first 3 h of culture and proceeds rapidly for at least the first 2 days of incubation. By this time 40%–50% of isotope usually has been released into the medium. Resident peritoneal macrophages appear to mobilize matrix as actively as those which are thioglycollate induced. By comparison, lymphocytes elicit little isotope mobilization from bone, and rat peritoneal exudate macrophages are markedly less efficient (P<0.001) at resorbing rat bone than are macrophages obtained from mice.Isotope release by peritoneal macrophages represents true cell-mediated resorption and not merely nonspecific mineral mobilization as evidenced by the facts that: (a) the magnitudes of release of isotopes representing the inorganic (45CaCl) and organic (3H-proline) phases of bone are the same, (b) daily buffering of the cultures to pH 7.4 has little effect on45Ca release, and (c) cell-matrix contact is required for optimal mobilization of45Ca or3H.

The response of human peripheral blood lymphocytes to phytohemagglutinin: Determination of cell numbers

Abstract Optimization and definition of conditions for studying lymphocyte function in vitro resulted in exponential proliferation of lymphocytes from day 2 to day 5 with an average doubling time of 20 hr. The number of cells in culture on day 5 was 5–10 times as great as the number initially planted and 10–20 times as great as the number surviving in culture on day 2. An improved pronase-cetrimide technique was used to determine the number of viable lymphocytes as a function of time after addition of PHA. The volume changes in nuclei, obtained after cetrimide treatment, were quantitated using a curve-fitting computer program. The response could be described in terms of an induction phase (0–2 days) characterized by a decrease in cellularity and an increase in nuclear volume, a proliferation phase (2–5 days) characterized by an exponential proliferation and a continued increase in the number of cells having a large nuclear volume, and a lysis phase (5–14 days) characterized by a decrease in cellularity and a decrease in nuclear volume. The results reported here suggest that the ratio of the number of cells cultured to the volume of culture medium was crucial for optimal transformation and proliferation, 105 cells/ml producing far better responses than 106 cells/ml.

DNA-dependent RNA polymerase levels during the response of human peripheral lymphocytes to phytohemagglutinin

The cellular levels of the various RNA polymerases have been monitored in resting human peripheral lymphocytes and in lymphocytes stimulated by phytohemagglutin. Activity was measured in the presence of exogenous templates following solubilization and chromatographic resolution of the different RNA polymerases. Resting lymphocytes contain Class I, II, and III RNA polymerases, although the respective levels of activity are very low compared to the levels in metabolically active cell types. During the PHA-induced transformation of resting lymphocytes, the Class I, II, and III enzyme levels rise dramatically. During four days exposure to PHA, the levels of RNA polymerases I and III (which synthesize, respectively, rRNA and the transfer and 5S RNAs) increase 17 fold, while the level of RNA polymerase II (which synthezies heterogeneous nuclear RNA) increase 8 fold. The possible relationship between enzyme levels and the regulation of gene expression is discussed.

Effect of Irradiation on Immune Responses

The radioresistance of antibody-mediated immune response is lowest when radiation is given before antigen, and rises steadily with time after antigen stimulation to reach a maximum when serum antibody is at a maximum, suggesting that antibody-producing cells are highly resistant. Irradiation affects the antibody-mediated response by 1) lengthening the period between stimulation and production of detectabel levels of serum antibody, 2) decreasing the rate of increase in serum antibody levls, and 3) decreasing the maximum concentration of serum antibody. Irradiation affects the cell-mediated response by inhibiting immunization and delayed hypersensitivity reactions when administered before antigen. Because the cell-mediated response involves nonlymphoid cells whose precursors are also radiosensitive, irradiation following stimulation may also produce temporary suppression.

Replication of lactic dehydrogenase virus and Sindbis virus in mouse peritoneal macrophages. Induction of interferon and phenotypic mixing

Abstract Mouse peritoneal macrophages stimulated by thioglycollate can proliferate in culture for over 2 wk. Sindbis virus replicates in these cells, but not in the initial exudate culture. Lactic dehydrogenase virus (LDV) also produces higher yields in the exponentially growing cells. Proliferating macrophages infected with LDV induce a factor that inhibits the replication of Sindbis virus. The factor was identified as interferon by its species specificity and its acid stability. Macrophages that have been in culture for only 24 hr do not produce interferon. Replication of Sindbis virus in LDV-infected macrophages leads to phenotypic mixing between the two virions. We have detected both pure pseudotypes—virions containing the RNA of Sindbis virus and the envelope of LDV—and particles containing the envelope proteins of both virions. Our results demonstrate that the restricted host range of LDV is not due to the inability of the virus to adsorb to cells other than macrophages.

Role of the regional lymph nodes in the cure of a murine lymphosarcoma

Using an experimental model with a transplanted murine lymphosarcoma, several observations strongly suggest that following tumor transplantation, there is a selective transient accumulation of tumor cells in the regional lymph nodes. Furthermore, the surgical removal of these lymph nodes immediately prior to or within a week following implantation of the tumor cells significantly impairs the cure of the tumor by a dose of local irradiation which in control animals cures 83%. Local irradiation of the regional lymph nodes (3000 rads single dose) produced a moderate decrease in cure rates, not as severe as that observed in the lymphadenectomized animals. It is postulated that there is a critical period of time during which an immune response is initiated in the regional lymph nodes, following antigen injection. If tumor cells have reached the regional lymph nodes and the immune response has been initiated by the time a tumor becomes clinically apparent, then prophylactic irradiation or dissection of these lymph nodes should not interfere with tumor control by the usual therapeutic methods. However, if this is not the case, and the body has not recognized the tumor antigens, then lymphadenectomy and to a much lesser degree, irradiation, may result in less effective therapeutic results. Further understanding of the processes involved in tumor dissemination in patients is necessary to correlate these experimental observations with the clinical management of cancer.

Macrophage growth inhibitors derived from the murine peritoneal cavity

SummaryThe murine peritoneal cavity contains factors that inhibit the in vitro growth and colony formation of macrophages. The inhibition of macrophage growth is not due to cell death. In the presence of inhibitors, the growth of colony-forming macrophages is suppressed, and small clusters are formed as a result of limited proliferation. The more mature mono-nuclear phagocytes (blood monocytes and peritoneal exudate macrophages) are more sensitive to the overall inhibitory effect of the peritoneal inhibitors than the less mature bone marrow mononuclear phagocytes. Furthermore, using dialysis and Amicon ultrafiltration, at least two inhibitors with differential inhibitory effects can be demonstrated. The colony formation of bone marrow mononuclear phagocytes is suppressed mainly by a protease-resistant, small molecular weight (<1,000) dialyzable inhibitor. In contrast, peritoneal exudate macrophages are sensitive to both the small molecular weight inhibitor and a protease-sensitive, large molecular weight (>12,000), nondialyzable inhibitor. The data suggest a possible existence of a dual inhibitor control on the proliferation of mononuclear phagocytes in vivo. In addition, the in vitro cultured peritoneal exudate cells are capable of producing inhibitors that mimic the activity of the in vivo inhibitors.

Lactic dehydrogenase virus replicates in somatic cell hybrids of mouse peritoneal macrophages and SV40-transformed human fibroblasts.

Abstract Hybrid clones obtained from mouse peritoneal macrophages and SV40-transformed human Lesch-Nyhan fibroblasts are permissive for the replication of lactic dehydrogenase virus. Immunofluorescent assays show that all of the cells are infected. These hybrid cells produce interferon in response to LDV infection as had previously been shown for LDV-infected macrophages.

Experimental observations on the significance of cell burden in tumor control

Experiments using a transplanted murine lymphosarcoma in syngeneic mice show that an initial inoculum of at least 106 tumor cells is needed to generate an immune response adequate to repel subsequent challenge from similar cells. The relationship among cell burden, dose of x radiation, and tumor control was studied. The experiments demonstrated that the number of tumor cells rejected as the result of an immunizing inoculation is approximately equivalent to the number of cells needed to initiate immunity. A correlation was established between the size of the initial injection and the probability of tumor invasion of regional lymph nodes. In addition, the existence was shown of a progressive increase in the number of tumor cells in axillary lymph nodes in mice in which the primary tumor recurs following irradiation, while in those mice in which the primary tumor is controlled, tumor cells in the axillary nodes disappear shortly after the local treatment. Several trials indicated that in this particular tumor system, the hosts immunologic response will prevent the growth of 104−105 cell inocula but that larger numbers of cells overwhelm the natural defenses and grow unabated. Our evidence supports the clinical observation that moderate doses of irradiation will control subclinical tumor cell populations. In addition, it is suggested that the immune response of the host may enhance the effects of radiation not only by increasing local cell kill but also by preventing the proliferation of disseminated cells outside the irradiated volume.

FACTORS AFFECTING THE MEASUREMENT AND INTERPRETATION OF IN VITRO LYMPHOCYTE REACTIVITY

Publisher Summary This chapter explores factors affecting the measurement and interpretation of in vitro lymphocyte reactivity. In a study described in the chapter, human peripheral blood lymphocytes were isolated using the Ficol–Hypaque procedure. After a wash in 50 ml of medium, the cells were appropriately adjusted and cultured in 13 × 100 mm glass tubes with or without 8 μg PHA (DIFCO P)/ml. Cultures were incubated in a humidified 10% CO 2 in air atmosphere at 37°C. Culture medium was α MEM supplemented with 10% fetal calf serum, 100 units penicillin, and 100 μg streptomycin/ml. Culture cellularity was determined daily for 11 days by the pronase–cetrimide technique. Lymphocytes were cultured at a variable concentration in 1 ml ranging from 2.5 to 200 × 10 4 cells/ml. When lymphocytes were cultured at an initial concentration of less than 2 × 10 5 cells/ml, they proliferated exponentially, with a doubling time of 20 h after an initial 48 h lag period during which about half the cells died. The length of time cells maintained this constant doubling time increased from day 5 for 10 5 cells/ml to day 7 for 25,000 cells/ml. The results obtained in this study suggest that medium becomes a limiting factor in cell proliferation when the cell concentration is increased.