Separation of functionally distinct subpopulations of Corynebacterium parvum-activated macrophages with predominantly stimulatory or suppressive effect on the cell-mediated cytotoxic T cell response.

Peritoneal cells (PEC) from mice injected ip with Corynebacterium parvum (CP) showed greatly enhanced suppressive activity on the growth of syngeneic tumor cells and on the generation of alloreactive cytotoxic T lymphocytes (CTL) in vitro. On the other hand, CP-activated PEC exhibited increased immunostimulatory (accessory or A cell) activity as measured by the restoration of the CTL response of nonadherent spleen cells. After fractionation of the CP-activated PEC according to cell size by velocity sedimentation, the mutually antagonistic A cell and immunosuppressive activities were clearly separated and found to be associated with functionally distinct subpopulations of macrophages. Thus A cell function was detected in fractions rich in small and medium sized macrophages which were probably derived from recently arrived monocytes. Immunosuppressive (and anti-tumor) activity was associated with the largest macrophages which were almost devoid of A cell function and probably represented a highly activated and differentiated macrophage subpopulation.     

Schizophyllan (SPG)-treated macrophages and anti-tumor activities against syngeneic and allogeneic tumor cells

We tested anti-tumor activities of macrophages treated with a neutral polysaccharide, schizophyllan (SPG), against syngeneic and allogeneic tumor cell lines. SPG was a macrophage stimulant which was not mitogenic to lymphocytes. That made a sharp contrast with the data that Corynebacterium parvum, BCG, and muramyl dipeptide (MDF) were macrophage stimulants which had lymphocyte-activating properties. Treatment of SPG-treated PEC with Thy12 monoclonal antibody and guinea pig complement did not affect the capabilities of tumor-cell-growth suppression by the treated PEC. Thus, the effector cells were peritoneal adherent cells (macrophages morphologically) and effector-to-target contact seemed to be necessary for effective tumor-cell-growth inhibition, although contradictory data exist for this. Murine peritoneal adherent cells harvested 4 days after a single IP injection of SPG at a dose of 100 mg/kg body weight of mouse showed the most prominent cytostatic and cytotoxic activities against syngeneic and allogeneic tumor cells. The distribution of anti-tumor activity in macrophages of various sizes followed the same pattern as macrophages treated with C. Parvum, i.e., larger macrophages showed more remarkable anti-tumor activity. Crude nonadherent peritoneal cells incubated with SPG at a concentration of 10 micrograms/ml, 100 micrograms/ml, or 1 mg/ml did not secrete lymphokine that rendered macrophages cytotoxic, while ConA-treated nonadherent cells did so. Furthermore, spleen cells treated with SPG in vivo did not secrete macrophage-activating lymphokine in the presence of SPG. On the other hand, addition of 1 mg/ml of SPG-treated peritoneal adherent cells and bone-marrow-derived macrophages in vitro rendered them cytotoxic to a moderate degree. This implies that SPG may activate macrophages directly, allowing them to become cytotoxic in the peritoneal cavity. Lastly, SPG could induce production of II-1-like factor to a moderate degree. SPG, whose molecular structure is well elucidated, will provide us with a strong tool to analyze the mechanism of macrophage activation both in vitro and in vivo.     

Nonspecific cytotoxicity of vaccinia-induced peritoneal exudates in hamsters is mediated by Thy-1.2 homologue-positive cells distinct from NK cells and macrophages

Vaccinia virus-induced peritoneal exudate cells (PEC) in the hamster were characterized with regard to cell type(s), target specificity, and expression of the T cell antigen, Thy 1.2 homologue. Hamsters were immunized intraperitoneally with vaccinia virus and cytotoxicity was measured against 51Cr-labeled targets in a 16-hr assay. PEC collected 4 days after immunization were cytotoxic for both baby hamster kidney cells (BHK) and herpes virus-infected BHK (BHKHSV). Both the nonadherent (lymphocyte) and adherent macrophage (MP) fractions of PEC were cytotoxic. Treatment of cells with a monoclonal anti-murine Thy 1.2 antibody (alpha-Thy 1.2) known to detect a Thy 1.2 homologue on hamster T cells, removed all of the cytotoxicity in both PEC fractions, whereas, cytotoxic spleen cells from the same animals were resistant to antibody treatment. Similarly, the cytotoxic cells in PEC induced by bacillus Calmette-Guérin were exclusively of the Thy 1.2 homologue-positive phenotype. Target specificities of Thy 1.2+ PEC and Thy 1.2- spleen cells were similar as evidenced by comparable activity against hamster BHK and BHKHSV targets and murine SV3T3 and YAC-1 targets. Previous studies have attributed the cytotoxicity of the adherent PEC to MP. However, as determined by immunofluorescence and morphological studies, treatments that enriched for MP decreased cytotoxic activity, whereas, procedures that enriched for lymphocytes enhanced cytotoxic activity suggesting that all cytotoxicity in PEC is mediated by a non-specific Thy 1.2 homologue positive lymphocyte (Thy 1.2+ CL). Thus our data support the conclusion that intraperitoneal inoculation of hamsters with vaccinia induces two distinctly compartmentalized phenotypes with similar cytotoxic characteristics--the Thy 1.2+ CL and the Thy 1.2 homologue-negative natural killer cell (NK) or NK-like cell in the peritoneum and in the spleen, respectively.     

Schizophyllan (SPG)-treated macrophages and anti-tumor activities against syngeneic and allogeneic tumor cells

Summary We tested anti-tumor activities of macrophages treated with a neutral polysaccharide, schizophyllan (SPG), against syngeneic and allogeneic tumor cell lines. SPG was a macrophage stimulant which was not mitogenic to lymphocytes. That made a sharp contrast with the data that Corynebacterium parvum, BCG, and muramyl dipeptide (MDF) were macrophage stimulants which had lymphocyte-activating properties. Treatment of SPG-treated PEC with Thy12 monoclonal antibody and guinea pig complement did not affect the capabilities of tumor-cell-growth suppression by the treated PEC. Thus, the effector cells were peritoneal adherent cells (macrophages morphologically) and effector-to-target contact seemed to be necessary for effective tumor-cell-growth inhibition, although contradictory data exist for this. Murine peritoneal adherent cells harvested 4 days after a single IP injection of SPG at a dose of 100 mg/kg body weight of mouse showed the most prominent cytostatic and cytotoxic activities against syngeneic and allogeneic tumor cells. The distribution of anti-tumor activity in macrophages of various sizes followed the same pattern as macrophages treated with C. Parvum, i.e., larger macrophages showed more remarkable anti-tumor activity. Crude nonadherent peritoneal cells incubated with SPG at a concentration of 10 g/ml, 100 g/ml, or 1 mg/ml did not secrete lymphokine that rendered macrophages cytotoxic, while ConA-treated nonadherent cells did so. Furthermore, spleen cells treated with SPG in vivo did not secrete macrophage-activating lymphokine in the presence of SPG. On the other hand, addition of 1 mg/ml of SPG-treated peritoneal adherent cells and bone-marrow-derived macrophages in vitro rendered them cytotoxic to a moderate degree. This implies that SPG may activate macrophages directly, allowing them to become cytotoxic in the peritoneal cavity. Lastly, SPG could induce production of II-1-like factor to a moderate degree. SPG, whose molecular structure is well elucidated, will provide us with a strong tool to analyze the mechanism of macrophage activation both in vitro and in vivo.Abbreviations PEC peritoneal exudate cells - SPG schizophyllan - LPS lipopolysaccharide - Con A concanavalin A - CGN carrageenan - B. M. bone marrow - FCS fetal calf serum - BCG bacille Calmétte Guérin - Il-1 interleukin 1 - PPD pure protein derivatives - MDP muramyl dipeptide - C. parvum Corynebacerium parvum Dr. Sugawara is a Research Fellow of the Alberta Heritage Foundation for Medical ResearchDr. Lee is a Research Associate of the National Cancer Institute of Canada     

Further studies on generation of macrophages in in vitro cultures of mouse spleen cells and its inhibition by the capsular polysaccharide of Klebsiella pneumoniae.

Although the number of macrophages detected in cultures of mouse spleen cells at the start of the culture was very small, it markedly increased during further incubation. Macrophages were generated not only from the glass-adherent cell fraction of spleen cells, but also from the nonadherent cell fraction obtained after removal of adherent cells either by incubating in glass petri dishes or by passing through a glass bead column. The generation of macrophages from the nonadherent cell fraction occurred even when it was separated as late as 48 hr after the start of the culture. The phagocytic activity of macrophages newly generated from the nonadherent cell fraction was relatively weak, but it was activated during further incubation. Based on these results, the maturation process of macrophages can be divided into at least the following four stages; glass-nonadherent nonphagocytic precursor cells, glass-adherent nonphagocytic precursor cells, immature macrophages with low phagocytic activity, and mature macrophages with full phagocytic activity. The addition of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) to cultures of spleen cells markedly suppressed the generation of macrophages. The suppressive effect of CPS-K depended on its dosage, and the minimum concentration of CPS-K showing a definite effect was 0.05 μg/ml. CPS-K inhibited further generation of macrophages in either the nonadherent or adherent cell fraction at any time after the start of the culture. The suppressive effect of CPS-K on the generation of macrophages could not be reversed by simple washing of spleen cells which had been kept in contact with CPS-K for 3 hr. There was no evidence which showed that CPS-K exhibited direct cytotoxic effects on spleen cells in the culture.     

Induced suppression of the efferent phase of contact sensitivity in rats

A study of the immunosuppressive systems of rats has been conducted with special attention to whether suppressor cells can be induced to down-regulate the efferent limb of contact sensitivity. Contact sensitivity (CS)1 was induced in DA rats 5 days after immunization with trinitrochlorobenzene (TNCB). Intravenous pretreatment of naive rats with TNP-coupled syngeneic spleen cells 7 days before sensitization suppressed the induction of CS by 60%. Suppression of the inductive phase of CS could be transferred adoptively into syngeneic rats with spleen cells of such tolerized animals. Cell fractionation studies showed the OX8+ (CD8) T cell population (cytotoxic/suppressor) was responsible for the suppression in the afferent phase of CS. Such cells were incapable of suppressing preexisting CS. To investigate whether suppression could be induced for the efferent phase, spleen and peritoneal exudate cells (PEC) from rats tolerized by administering TNP-spleen cells iv plus epidermal paintings with TNCB were adoptively transferred into recipients sensitized 4 days earlier. Both spleen cells and PEC suppressed the efferent phase of CS but PEC did so more efficiently. Separation of splenic cells revealed the suppressors to be CD8+ T cells. Furthermore, separation of PEC into plastic adherent and nonadherent cells showed the nonadherent (T cell enriched) cells to be noneffective alone. The adherent subpopulation conveyed suppression but did so more effectively upon addition of the T cells. Thus, T cells and macrophages may operate in concert to achieve suppression of the efferent limb of CS. PEC from tolerized rats suppressed performed CS of any specificity but only after the suppressor cells were triggered with the same antigen that induced them. Since both the afferent and efferent phases of CS have now been shown to be suppressable, two separate suppressor mechanisms may be operable in rats.     

Antigen-specific augmentation of delayed-type hypersensitivity by immune serum factor in mice: augmentation of anti-tumor cytostatic activity

A humoral factor capable of augmenting delayed-type hypersensitivity antigen specificity (DAF) is present in the serum of mice sensitized with heterologous erythrocytes to induce a delayed footpad reaction (DFR). In the present study, a similar factor was identified when xenogeneic tumor cells were used as antigens. This factor also could augment the in vitro anti-tumor cytostatic activity against homologous tumor cells, which correlated with in vivo DFR to the same tumor cells. The cytostatic activity augmented by the transfer of this factor had the following characteristics: The activity appeared in the whole peritoneal exudate cells (PEC) from serum recipients at 4 days after the antigenic challenge. Such an activity was revealed in the collaboration of plastic dish-nonadherent and -adherent PEC as the primary and final effectors, respectively. The appearance of primary effector cells for such an activity was also accelerated in spleen and lymph node cells. However, a sufficient number of macrophages were always required as the final effectors in their functional expression. These primary effectors were sensitized T lymphocytes which produced lymphokine(s) such as macrophage-activating factor(s) and which contributed to this augmented cytostatic activity through the activation of macrophages. Thus, this immune serum factor seems to exert functional expression by accelerating the generation of lymphokine-producing delayed-type T lymphocytes, which is also responsible for cytostatic anti-tumor immunity.     

Suppressor T cells regulate the cytolytic T lymphocyte response to syngeneic tumors induced by murine sarcoma virus (MSV) in the mouse.

Studies were designed to analyze the immune activities of spleen cells from mice previously injected with murine sarcoma virus (MSV) and undergoing the processes of MSV tumor growth and rejection. Fractionation of MSV-primed spleen cells according to cell size by velocity sedimentation at unit gravity showed that MSV-specific cytolytic T lymphocytes (CTL) generated in vivo underwent an apparent transition in size from large to small cells as the tumor regressed. The majority of CTL precursors, however, were invariably recovered among small to medium-sized MSV-immune cells, as revealed to CTL generation in vitro in secondary mixed leukocyte-tumor cell cultures (MLTC). Evidence was obtained for the existence in MSV-immune spleens of two suppressor cell populations capable of inhibiting CTL generation in vitro: one population probably consisted of macrophages and could be removed by treatment with carbonyl iron; the second population was comprised of T cells and inhibited the differentiation of tumor-immune CTL precursors in a selective manner. These results provide a preliminary overview of the mechanisms regulating the generation, differentiation, and activity of tumor-specific CTL in a syngeneic model system.     

Effects of adriamycin on the activity of mouse natural killer cells

Adriamycin, a widely employed anti-neoplastic agent, was found to have either inhibitory or stimulatory effects on NK activity, depending on the site examined. A single i.p. administration of ADM resulted in a rapid increase of cytolytic activity by PEC of various mouse strains. The effector cells appeared to be NK cells, being nonadherent and nonphagocytic; they expressed low amounts of Thy 1.2 antigen and had the same pattern of specificity as splenic NK cells. In contrast to the stimulatory effects of NK activity of PEC, ADM caused a transient dose-dependent depression of NK activity in the spleen, with a peak reduction at day 3 and recovery within a few days thereafter. The depressed NK activity could be reversed by removal of adherent cells by passage through a nylon column. Moreover, ADM induced cytostatic activity against tumor cells by macrophages, suggesting that activated macrophages may be responsible for suppression of splenic NK activity. The possible modulation of the levels of NK activity by ADM-induced macrophages was supported by mixture experiments, in which plastic adherent spleen cells from ADM-treated mice, but not from normal mice, inhibited the NK activity of normal spleen cells.     

The role of the macrophage as the stimulator cell in contact sensitivity.

In this study we examined the requirement for the type of stimulator cell for thymus-derived (T) lymphocyte activation to simple chemical haptens. T cells from picryl chloride-immune guinea pigs were challenged in vitro with various trinitrophenyl (TNP)-conjugated syngeneic stimulator cells and the extent of activation was determined by an increase in DNA synthesis. Hapten-specific T cell activation occurred with TNP-conjugated peritoneal exudate cells (PEC) and purified macrophages but not with TNP-conjugated erythrocytes, thymocytes, or nonadherent lymph node cells or PEC. In addition, T cell activation also occurred with TNP-conjugated guinea pig leukemia cells, but only in the presence of macrophages. Furthermore, it was shown that macrophages were required to process and/or present TNP-conjugated leukemia cell antigens rather than simply providing a growth-promoting function. These results suggest that a macrophage-like stimulator cell is required for hapten-specific T cell activation and that this particular stimulator cell may be important in contact sensitivity.     

Phagocytic killing of Candida albicans by different murine effector cells

Three major phagocytic populations in the mouse were tested in vitro for killing of Candida albicans by means of 51Cr release assay: early inflammatory peritoneal polymorphonuclear cells (PMN), unfractionated or adherent spleen cells and resident peritoneal macrophages (PEC). Considerable candidacidal activity was found in the early inflammatory neutrophil and adherent spleen cell populations. On the contrary, only limited activity was found to be associated with resident peritoneal macrophages. The phagocytic killing apparently involved multiple mechanisms.     

Induction of interferon production in mouse spleen cell cultures by corynebacterium parvum.

Spleen cells of CS7BL/6 mice produced considerable amounts of interferon (IF) in vitro when tested 5 to 20 days after injection of killed Corynebacterium parvum. Interferon was also produced when C. parvum was added in vitro to spleen cell cultures of previously untreated mice. High levels were detected after 1 day of culture with some increment during subsequent days. In a number of experiments IF was also produced in untreated control cultures but only after prolonged cultivation and not after 1 day. The highest levels of IF were usually obtained when spleen cells of C. parvum-treated mice were challenged with additional C. parvum in vitro. The IF induced by C. parvum shared certain physicochemical properties with a tested immune IF and was not neutralized by an antiserum raised against a type I IF. Spleen cells of nu/nu mice and spleen cells treated by anti-θ serum plus complement did not differ from their respective controls, indicating that production of IF did not require mature T lymphocytes. Removal of B lymphocytes by nylon wool columns abolished the capacity of spleen cells to produce IF. When spleen cells were freed of adherent cells by the use of plastic surfaces, they no longer produced IF. Peritoneal exudate macrophages (PEC), which by themselves did not produce IF, in small numbers reconstituted nonadherent spleen cells. Nylon column-treated spleen cells, however, could not be restored by PEC. It is concluded that IF upon challenge with C. parvum is produced by B lymphocytes and requires the help of macrophages.     

Effect of concanavalin A treatment on the allogeneic response of mice to challenge with P 815 mastocytoma: interleukin 2 treatment reverses concanavalin A suppression in vivo

Mice injected repeatedly with concanavalin A (Con A) prior to and following challenge with P 815 mastocytoma are suppressed in their cell-mediated cytotoxicity responses. Earlier studies showed that pretreatment of the animals with silica to affect macrophage (M phi) functions reversed the Con A suppression. In the present paper we have shown that peritoneal exudate cells (PEC) induced/activated by ip injection of Con A were able to transfer suppression to normal mice. Separation of the PEC populations into adherent and nonadherent cells abrogated their capacity to transfer suppression. It was further shown that Con A is not functioning in this in vivo system to block effector activity of cytotoxic cells on target cells, and PEC induced with Con A were not directly cytotoxic to target P 815 cells. Finally, we were able to show that the cytotoxicity response of Con A-suppressed mice could be enhanced by treatment with concentrated culture supernatants of normal mouse spleen cells, rich in interleukin 2 (IL 2) activity. Attempts to detect a recently described mouse serum inhibitor of IL 2 in normal or Con A-treated mice were unsuccessful and spleen cells from Con A-treated mice lost their capacity to generate IL 2 in vitro when cultured under appropriate conditions. Taken together, these results suggest that suppression of cell-mediated immune responses in Con A-treated mice results from interruption of the normal generation of IL 2 helper effects necessary for the activation of cytotoxic effector T cells in vivo.     

Anti-tumor cytostatic mechanism and delayed-type hypersensitivity against a syngeneic murine tumor. Comparison between neonatally thymectomized mice and congenitally athymic nude mice

We studied the anti-tumor mechanism against a syngeneic tumor using a BALB/c-MA tumor system by cytolysis and cytostasis assays in vitro comparing mice neonatally thymectomized at 1 day or 7 days after birth (NTx-1, NTx-7), sham-operated (sham) mice, and congenitally athymic nude BALB/c mice. NTx-1 mice showed more rapid tumor growth and a slightly lower degree of strong cytostatic activity in peritoneal exudate cells (PEC) than NTx-7 or sham mice. Nude mice showed more rapid MA growth than NTx-1 mice and no cytostatic activity in PEC. After immunization with mitomycin C-treated MA (MMC-MA), NTx-1 mice acquired an immunoprophylactic capacity against MA and showed cytostatic activity and delayed footpad reaction (DFR) to MA, however, nude mice showed no acquisition of such an immunity, or cytostatic activity, or DFR to MA. These differences between NTx-1 and nude mice could be well-explained by less capacity of nude mice to produce a macrophage-activating factor, which activates macrophages to exert cytostasis and DFR. However, NTx-1 mice could not reject MA by immunization with MMC-MA in CFA (MMC-MA/CFA), although such immunized sham mice could eliminate MA completely. Both PEC and spleen cells from Sham mice immunized with MMC-MA/CFA showed cytostatic activity, whereas NTx-1 mice showed cytostatic activity of the same level in PEC and less in spleen cells compared to Sham mice. Cytolytic activity was never detected throughout this study in a BALB/c-MA system. These data suggest that cytostasis plays an important role in antitumor immunity against a syngeneic MA tumor and that two types of cytostasis is included from the standpoint of thymus-dependency of ontogenic development, relatively low and high.     

In vitro cytotoxicity of peritoneal macrophages activated with Mycobacterium smegmatis

Incorporation of 3H-TdR into EL4 leukemic cells in vitro was inhibited by peritoneal exudate cells (PEC) harvested from syngeneic C57BL/6J mice given an intraperitoneal (i.p.) injection of 1x10(7) viable Mycobacterium smegmatis ATCC 607 (Smeg) 4 days before. This phenomenon was also observed in the following five systems of PEC from animals and syngeneic tumor cells: C57BL/6J mice and B16 melanoma; DBA/2 mice and P815 mastocytoma; SWM/Ms mice and K5 fibrosarcoma; BALB/c, nu/nu mice and KKN-1 fibrosarcoma; and strain 2 guinea pigs and line-10 hepatoma. The in vitro cytotoxicity of the PEC activated by viable Smeg was much higher than those activated by dead-Smeg, viable BCG or proteose peptone. The activity of the adherent fraction of the PEC was stronger than that of the nonadherent one, and not influenced by either anti-theta or anti-mouse lymphocyte rabbit sera. The PEC induced with Smeg 4 days before contained a large population of mononuclear cells (88.9%) and a significant level of polymorphonuclear cells (PMN) (3.2%), and showed a much higher cytotoxicity than the PEC induced with Smeg 3 hr before, which contained a much larger population of PMN (71.9%), suggesting that PMN were not the effector cells in this system. In vitro and in vivo treatment with macrophage-inhibitors such as carrageenan, trypan blue and cytochalacin B, reduced the activity of the PEC. All of these facts suggested macrophages as the effector. Viable macrophages were required for the growth inhibition of EL4 in vitro: gamma-ray irradiated or freeze-thawed macrophages were ineffective. Kinetic studies revealed that inhibition of 3H-TdR incorporation into EL4 cells started within 3 hr of incubation together with the activated macrophages at an effector to target (E/T) ratio of 5, and the incorporation decreased gradually with the lapse of incubation time. On the other hand, 51Cr release from labelled EL4 was undetected when the E/T ratio was 5 but detected at on E/T of 10 or more. Even at the higher E/T ratio, at least 10 hr were needed until the release of 51Cr, suggesting that the activated macrophages produced growth inhibition of tumor cells followed by cell destruction.     

Mechanism(s) of in vitro macrophage activation with Nocardia rubra cell wall skeleton: the effects on macrophage activating factor production by lymphocytes

Summary BALB/c mouse peritoneal macrophages prepared from WPC which had been treated with N. CWS demonstrated potent cytostatic activity against syngeneic Meth A fibrosarcoma cells. The maximum cytostatic activity developed in the macrophages when WPC were incubated with 25 g/ml N. CWS for 3 days. NAPC from BALB/c mice given an i. p. injection with 100 g N. CWS 7 days previously (N. CWS-NAPC) or supernatants from N. CWS-NAPC also activated peritoneal macrophages in vitro. However, when peritoneal macrophages were incubated with N. CWS in the absence of NAPC, or when T cells were depleted from WPC by treatment with anti-Thy 1.2 antibody and complement, N. CWS failed to enhance the cytostatic activity of the macrophages. Furthermore, thioglycollate-elicited peritoneal macrophages from C3H/HeN mice increased their cytolytic properties by incubation with supernatant fluids from N. CWS-treated spleen cells. These findings suggest that in vitro macrophage activation with N. CWS depends on MAF secreted from T lymphocytes. Abbreviations used: N. CWS, Nocardia rubra cell-wall skeleton; BRM, biological response modifier; MAF, macrophage activating factor; IL-1, interleukin 1; IL-2, interleukin 2; IFN-, interferon gamma; PCCM, peritoneal cell culture medium; SCCM, spleen cell culture medium; TCM, tumor culture medium; HI-FCS, heat-inactivated fetal calf serum; Con A, concanavalin A; PC, peritoneal cells; PEC, peritoneal exudate cells; WPC, whole peritoneal cells; APC, adherent peritoneal cells; TGC-APC, thioglycollateelicited adherent peritoneal cells; NAPC, nonadherent peritoneal cells; SN, supernatants; NK cells, natural killer cells; LAK cells, lymphokine activated killer cells E:T ratio, effector: target cell ratio; WSA, water soluble adjuvant; LPS, lipopolysaccharide; MDP, N-acetyl-muramyl-L-alanyl-D-isoglutamine     

Macrophages suppress CTL generation in rat mixed leukocyte cultures.

The generation of CTL in rat MLC was actively suppressed by a cell population present in spleen cell preparations from normal rats. These suppressor cells were characterized by using a variety of cell fractionation techniques. Suppressor cells were removed by passage of spleen cells through nylon wool columns or by treatment with carbonyl iron. Suppressive activity was present in the mononuclear cell fraction of spleen cells obtained by Ficoll-Hypaque density gradient centrifugation. After velocity sedimentation at unit gravity, enrichment of suppressive activity was demonstrated in the fractions containing large cells as compared to the fractions containing small cells. Populations rich in macrophages were shown to have similar suppressive activity upon CTL induction in MLC. These studies suggest that macrophages present in normal rat spleen cell preparations account for the difficulty in generating CTL in MLC prepared with rat cells.     

Adriamycin-induced activation of NK activity may initially involve LAF production

C3HeB/FeJ spleen cells (unseparated or passaged over nylon wool columns) were cultured overnight (1-2 X 10(6) cells/microwell) in the presence and absence of resident or ADM-induced PEC and anti-YAC-1 (4h) NK activity was determined. The addition of resident PEC to spleen cells had little effect on NK activity. However, the addition of ADM-elicited PEC (10 mg/kg, IP, day -1 and day -5) to spleen cells prior to culture significantly augmented NK activity. If ADM-induced PEC were treated with carbonyl iron prior to coculture with spleen cells, augmentation of anti-YAC-1 activity was not observed. This suggested that ADM-activated macrophages augmented cultured splenic NK activity. Supernatants from overnight-cultured resident or ADM-induced adherent PEC were then prepared, dialyzed (to remove ADM), and tested for mitogenic activity or cocultured with spleen cells overnight. ADM-induced adherent PEC supernatants stimulated the proliferation of murine thymocytes (both LAF and IL-2 also stimulate) but not cultured CTL (only IL-2 stimulates). ADM-induced adherent PEC supernatants (as well as LAF, IL-2, and IFN) augmented overnight-cultured C3HeB/FeJ splenic NK activity. However, only IL-2 and IFN could augment overnight-cultured athymic BALB/c . nu/nu splenic NK activity. This suggested that ADM-elicited macrophages produce LAF which may act directly on NK cells or, more likely, may induce T cells to produce IL-2, IFN, or both.     

Synergistic cytolytic activity by combined populations of peritoneal T lymphocytes and macrophages during the L5178Y cell tumor-dormant state in DBA/2 mice

It was previously reported that the establishment of the L5178Y cell tumor-dormant state in DBA/2 mice is mediated principally by a peritoneal cytolytic T-cell response that reaches peak levels 4 days after L5178Y cell challenge, lyses more than 99% but less than 100% of peritoneal L5178Y cells, and gradually wanes to background levels by 40–70 days postchallenge (DPC). At this time the majority of mice are clinically normal, and contain a relatively small number of L5178Y cells in the peritoneal cavity. During the tumor-dormant state, mice that harbor more than 10⁴ L5178Y cells contain peritoneal macrophage-mediated cytolytic activity. We report here that tumor-dormant mice that contain fewer than 10⁴ peritoneal L5178Y cells also produce cytolytic activity in vitro, but that it is synergistic, in that the cytolytic activity of adherent (AD) peritoneal cells (PEC) and nonadherent (NAD) PEC cultured together is greater than the additive lysis produced by these cell populations when cultured separately. This synergistic cytolytic activity is: (1) effector cell density dependent, (2) dependent on the tumor-dormant status of the NAD and AD PEC donor mice, (3) protracted in its kinetics during a 48-hr in vitro assay, and (4) dependent on an interaction between NAD T cells and AD phagocytic macrophages. The consistent detection of this in vitro-assayed cytolytic activity in PEC of tumor-dormant mice which harbor small endogenous tumor burdens suggests that it reflects an in vivo cytotoxic effector mechanism involved in the long-term maintenance of the tumor-dormant state.     

Antibody-dependent murine macrophage-mediated damage to Schistosoma mansoni schistosomula in vitro

Antibody-dependent cell-mediated cytotoxicity (ADCC) against schistosomula of the human parasite Schistosoma mansoni was demonstrated using antisera from mice plus peritoneal exudate cells (PEC). PEC were divided into plastic-adherent (96% macrophages, 4% lymphocytes) and nonadherent (92% lymphocytes, 8% macrophages) cell populations. Four criteria of ADCC were used, including minimal and maximal cell attachment, and death of and uptake of trypan blue by schistosomula. Using cells from normal mice and antisera from schistosome-infected mice, macrophages adhered to, damaged the tegument and underlying structures of, and killed schistosomula when observed following 18 hr incubation. In homologous systems, the results were similar when outbred CD-1 and inbred BALB/c mice were compared, except that potency of antisera from the latter mice decreased after 6–7 weeks postinfection, whereas the opposite was true for the former strain of mice. Nonadherent cells also exhibited ADCC against schistosomula, but the potency was considerably lower than that of adherent cells. These complement-independent ADCC reactions were stage-specific for the schistosomulum in that no reactions occurred with adult worms.