Peritoneal Exudate Cells Cells Research Articles

Modulatory Effect of Killed Propionibacterium acnes and Its Purified Soluble Polysaccharide on Peritoneal Exudate Cells from C57Bl/6 Mice: Major NKT Cell Recruitment and Increased Cytotoxicity

Propionibacterium acnes has been described as a potent adjuvant to immune responses in vitro and in vivo. Presently, we analysed the modulation of peritoneal exudate cells (PEC) by heat-killed P. acnes or its purified soluble polysaccharide (PS), both injected intraperitoneally in C57Bl/6 mice, aiming at their recruitment and cytotoxicity. Both treatments induced an increase in macrophages, immature dendritic cells, B1a lymphocytes and NK1.1(+) CD3(+) cells. The bacterium caused a remarkable increase in a NK1.1(+) CD3(+) CD4(-) CD8(-) cells subpopulation, whereas the PS component seemed responsible for the recruitment of mainly macrophage cells. To assess P. acnes and PS adjuvant effect on PEC cytotoxicity we evaluated their in vitro effect on murine B16F10 melanoma cells. The effector cells from the heat-killed bacteria and PS-treated groups lysed melanoma cells in co-cultures with PEC. Mice genetically deficient in IFN-gamma, when stimulated with P. acnes or PS, had reduced PEC cytotoxicity, and the cytotoxic effect was completely abrogated in PEC from iNOS(-/-) mice. The tumoricidal activity of PEC from P. acnes-treated mice was mediated by macrophages and NKT cells stimulated with IL-12. In PS-treated mice the cytotoxicity was mediated mainly by macrophages. Moreover, both treatments increased IL-4 and IFN-gamma production by NKT cells. In conclusion, we show that P. acnes act mainly by recruiting and activating NKT double-negative cells in PEC, which were shown to be tumoricidal in vitro when induced by IL-12. Macrophages induced by both P. acnes and PS have their antitumour effect dependent on NO production.

Role of IFN-gamma on dissociation between nitric oxide and TNF/IL-6 production by murine peritoneal cells after restimulation with bacterial lipopolysaccharide.

Murine peritoneal exudate cells (PEC) pre-exposed to bacterial lipopolysaccharide (LPS) show augmented nitric oxide (NO) production by LPS restimulation, in contrast to LPS tolerance with reduced production of tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6). Significant amounts of interferon-gamma (IFN-gamma) were detected in the PEC cultures on LPS stimulation, and anti-IFN-gamma antibody suppressed the LPS-induced NO, but not TNF-alpha and IL-6, production. Addition of anti-IFN-gamma antibody to the cultures in the LPS pre-exposure step strongly suppressed the augmented NO production on LPS restimulation. Anti-IL-12 antibody, which suppressed the LPS-induced IFN-gamma production, also suppressed the augmented NO production, as did anti-IFN-gamma antibody. Taken together, we propose the following mechanisms: (1) T and NK cells in PEC produce IFN-gamma by the action of IL-12, which is derived from LPS-stimulated macrophages, and (2) the de novo-produced IFN-gamma activates macrophages to augment NO production on LPS restimulation.

Macrophages are the major reservoir of latent murine gammaherpesvirus 68 in peritoneal cells.

B cells have previously been identified as the major hematopoietic cell type harboring latent gammaherpesvirus 68 (gammaHV68) (N. P. Sunil-Chandra, S. Efstathiou, and A. A. Nash, J. Gen. Virol. 73:3275-3279, 1992). However, we have shown that gammaHV68 efficiently establishes latency in B-cell-deficient mice (K. E. Weck, M. L. Barkon, L. I. Yoo, S. H. Speck, and H. W. Virgin, J. Virol. 70:6775-6780, 1996), demonstrating that B cells are not required for gammaHV68 latency. To understand this dichotomy, we determined whether hematopoietic cell types, in addition to B cells, carry latent gammaHV68. We observed a high frequency of cells that reactivate latent gammaHV68 in peritoneal exudate cells (PECs) derived from both B-cell-deficient and normal C57BL/6 mice. PECs were composed primarily of macrophages in B-cell-deficient mice and of macrophages plus B cells in normal C57BL/6 mice. To determine which cells in PECs from C57BL/6 mice carry latent gammaHV68, we developed a limiting-dilution PCR assay to quantitate the frequency of cells carrying the gammaHV68 genome in fluorescence-activated cell sorter-purified cell populations. We also quantitated the contribution of individual cell populations to the total frequency of cells carrying latent gammaHV68. At early times after infection, the frequency of PECs that reactivated gammaHV68 correlated very closely with the frequency of PECs carrying the gammaHV68 genome, validating measurement of the frequency of viral-genome-positive cells as a measure of latency in this cell population. F4/80-positive macrophage-enriched, lymphocyte-depleted PECs harbored most of the gammaHV68 genome and efficiently reactivated gammaHV68, while CD19-positive, B-cell-enriched PECs harbored about a 10-fold lower frequency of gammaHV68 genome-positive cells. CD4-positive, T-cell-enriched PECs contained only a very low frequency of gammaHV68 genome-positive cells, consistent with previous analyses indicating that T cells are not a reservoir for gammaHV68 latency (N. P. Sunil-Chandra, S. Efstathiou, and A. A. Nash, J. Gen. Virol. 73:3275-3279, 1992). Since macrophages are bone marrow derived, we determined whether elicitation of a large inflammatory response in the peritoneum would recruit additional latent cells into the peritoneum. Thioglycolate inoculation increased the total number of PECs by about 20-fold but did not affect the frequency of cells that reactivate gammaHV68, consistent with a bone marrow reservoir for latent gammaHV68. These experiments demonstrate gammaHV68 latency in two different hematopoietic cell types, F4/80-positive macrophages and CD19-positive B cells, and argue for a bone marrow reservoir for latent gammaHV68.

Peritoneal macrophages play an important role in eliminating human cells from severe combined immunodeficient mice transplanted with human peripheral blood lymphocytes.

To elucidate the mechanism of human cell elimination from severe combined immunodeficient (SCID) mice transplanted with human peripheral blood lymphocytes (hu-PBL-SCID mice), we explored the immunocytes in the peritoneal cavity in SCID mice where human PBL were transferred. When the phenotype of peritoneal exudate cells (PEC) was compared by flow cytometry among three congenic strains of SCID mice that differ in their acceptability for human PBL, the PEC in NOD-scid mice, which exhibit the highest acceptability, contained the smallest number of F4/80lo/-Mac-1(+)-activated macrophages. Moreover, the proportions of natural killer cells in PEC of the three strains of SCID mice were not always correlated with the acceptability. These findings suggest the possibility that peritoneal macrophages eliminate human cells in hu-PBL-SCID mice. To verify this hypothesis, we evaluated the engraftment of human PBL into SCID mice that were treated with liposome-encapsulated dichloromethylene diphosphonate, which selectively depletes macrophages by inducing apoptosis, or 8-aminoguanidine hemisulphate salt, an inhibitor of inducible nitric oxide synthase of macrophages. As a result, both of these regimens improved engraftment of human PBL, indicating that peritoneal macrophages take part in human cell elimination in the peritoneal cavity of hu-PBL-SCID mice and that it is mediated, at least in part, by direct macrophage cytotoxicity utilizing nitric oxide.

Increase of heat-shock protein and induction of gamma/delta T cells in peritoneal exudate of mice after injection of live Fusobacterium nucleatum.

Fusobacterium nucleatum and Actinobacillus actinomycetemcomitans are Gram-negative rod periodontal pathogens. The peritoneal cavity of Institute of Cancer Research (ICR) mice was used as the local infection model. In vivo production of heat-shock proteins (hsp) was studied by injection of 1/10 minimum lethal dose (MLD) of each live bacteria into mice. Heat-shock proteins 70 and 60 were examined in the extract of peritoneal exudate cells (PEC) from mice injected intraperitoneally with either F. nucleatum or A. actinomycetemcomitans by using sodium dodecylsulphate-polyacrylamide gel electrophoresis and immunoblotting analysis. Although hsp are present in PEC without injection of the bacteria, both hsp increased and reached a peak on day 3 after F. nucleatum injection but not after A. actinomycetemcomitans. Kinetic study of gamma/delta cells in PEC after injection of bacteria showed that the increase of gamma/delta T cells was observed only in the PEC from mice injected with F. nucleatum but not A. actinomycetemcomitans. The gamma/delta T cells in PEC were either CD3+ and CD4+ or CD3+ and CD8+. The differential cell count of PEC suggested that gamma/delta T-cell induction is related to the expansion of the macrophage population. The phagocytic and chemiluminescence responses of macrophages against the same bacteria were compared after intensive immunization with live F. nucleatum and A. actinomycetemcomitans. Elevations of chemiluminescence response and phagocytic function by immunization were observed in the macrophages of mice immunized with F. nucleatum. These results suggest the sequential appearance of hsp, gamma/delta T cells and macrophage activation after fusobacterial infection.

Immunomodulatory activity of a novel nucleoside, 7-thia-8-oxoguanosine: I. Activation of natural killer cells in mice.

We reported recently that a novel immunomodulator, 7-thia-8-oxoguanosine (7T8OG)2 inhibited formation of pulmonary melanoma metastases (1), prevented against viral infection in mice (2) and potentiated the efficacy of a weakly immunogenic leukemia vaccine (3). Since certain tumor metastases and virus infected cells are targets to natural killer cells (NK cells), we now investigated whether 7T8OG is capable of activating NK cells in mice using NK cell sensitive YAC-1 and B16 and NK cell insensitive P815 targets. CBA/CaJ spleen cells incubated in vitro with 7T8OG at concentrations ranging from 0.005 to 0.5 mM responded with increased NK cell activity (32-62%) compared to controls (4-8%) to YAC-1 targets. Similar levels of augmentation in NK cell activity were observed when 40-168 mg/kg of 7T8OG was administered in vivo. In addition to the spleen, 7T8OG activated NK cells in the bone marrow (BM), the lungs, the liver, and in peritoneal exudate cells (PE). Although 7T8OG elicited activation of NK cells was observed as early as three hours after treatment, the maximal activity was observed after 24 h in the spleen; 12 h in the BM; 48 h in the lungs, and 72 h in PE. Administration of the drug by s.c., i.v., and i.p. routes all induced activation of NK cells in spleen, BM and PE. 7T8OG was found to activate NK cells in seven inbred and an outbred mouse strain, suggesting that the induced cytotoxicity against allogeneic and syngeneic tumor cells is not strain specific as well as independent of MHC restriction. C3H/He, CBA/CaJ and BDF/1 displayed higher levels of increased NK cell activity, whereas AKR mice were low responders. Low concentrations of IL-2 (0.25-5 U/ml) that induce little or no NK cell activity, when used in combination with 7T8OG, elicited significant enhancement of NK cell cytotoxicity. In contrast, IFN and 7T8OG showed no such synergism.

Down‐regulation of adoptive adjuvant‐induced arthritis by suppressor factor(s)

We recently described a method for inducing immunologic tolerance to trinitrochlorobenzene (TNCB), a hapten that generates suppressor cells capable of down-regulating the efferent phase of TNP-specific contact hypersensitivity in rats. Peritoneal exudate cells (PEC) of such tolerized rats, upon being triggered by specific hapten, suppressed contact hypersensitivity to another hapten elicited at the same time. This implied that cells that mediate delayed-type hypersensitivity of any specificity might be down-regulated, provided that the suppressor cells are activated with specific antigen and that the unrelated delayed-type hypersensitivity is elicited in parallel. To rigorously test this possibility, we examined the ability of TNP-specific suppressor lymphoid cell factors to affect cells that mediate adoptively transferable adjuvant-induced arthritis (AIA) in rats. To induce arthritis, spleen cells from Freund's complete adjuvant-injected rats were stimulated with concanavalin A and administered to naive recipients. Prior to adoptive transfer, the cells were exposed for brief intervals to supernatants of lymphoid cells from control and hapten-tolerized rats. Supernatants of PEC and lymph node cells from hapten-tolerized rats were found to markedly reduce the effectiveness of cells that mediate AIA. The hapten-tolerized cells required reexposure to hapten prior to preparation of the supernatants. Supernatants of spleen cells from hapten-tolerized rats that had been hapten-painted as well as hapten-triggered and supernatants of lymph node cells and of PEC from only hapten-painted or hapten-triggered rats were ineffective in altering the AIA. Thus, factors from suppressor cells induced toward hapten-coupled self-antigens have been found to adversely affect the function of lymphoid cells that mediate a totally unrelated inflammatory response, namely, AIA. The clinical implications of these findings are discussed.

The in vivo Anti‐tumor Effect of Human Recombinant Interleukin‐6

Administration of recombinant interleukin‐6 (IL‐6) was found to induce in vivo generation of cytotoxic T lymphocytes (CTL) against syngeneic transplantable erythroleukemia (FBL‐3) in lymph node cells and peritoneal exudate cells (PEC) in C57BL/6 mice. Furthermore, 15 out of 16 C57BL/6 mice injected with 5 × 106 viable FBL‐3 cells survived on day 100 when they were treated with 5 × 104 U of recombinant IL‐6 three times a day on days 1, 2, 3, 5, 7 and 9 after the inoculation of tumor cells (the cure rate was 94%). Cured mice could reject the tumor cells rapidly after the re‐inoculation of a large number of live FBL‐3 cells. In contrast, all normal mice died of tumor development by day 10. In these cured mice, FBL‐3‐specific CD4‐8+ CTL cells were found to be generated in PEC, spleen and lymph node cells by either in vivo or in vitro re‐stimulation with FBL‐3 cells, but lymphokine‐activated killer cells never developed. The results suggested that the anti‐tumor effect of IL‐6 was mediated by in vivo induction of tumor‐specific CTL.

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.

Pronounced antitumor effect of LAK-like cells induced in the peritoneal cavity of mice after intraperitoneal injection of OK-432, a killed streptococcal preparation.

More than 80% of BALB/c mice bearing BAMC-1 ascites tumor were completely cured after five consecutive (once every 2 days) i.p. injections of a 0.1 mg dose of OK-432, beginning on day 2 after tumor implantation. The antitumor effect of OK-432 was abolished in athymic nu/nu mice and in anti-thymocyte globulin-treated euthymic BALB/c mice, so although OK-432 treatment did increase the length of survival, all animals eventually died as a result of tumor growth. When peritoneal exudate cells (PEC), obtained on day 12 from OK-432-treated BAMC-1-bearing euthymic mice were evaluated for in vivo tumor neutralization activity, all mice receiving an i.p. injection of the admixture of the nonadherent PEC (1 X 10(7) cells) with BAMC-1 cells (1 X 10(5)) survived for more than 60 days. When the same nonadherent PEC (1 X 10(7) cells) were i.p. transferred adoptively 1 day after the inoculation of 1 X 10(5) BAMC-1 tumor cells, again all mice survived. When these in vivo active PEC were tested for cytotoxicity in vitro against fresh BAMC-1 tumor cells, natural killer (NK) sensitive syngeneic RL male 1, NK-sensitive allogeneic YAC-1 cells, NK-resistant syngeneic Meth-A cells, allogeneic tumor cells (EL4, B16, and P815) and xenogenic human cells, the PEC were found to be capable of lysing BAMC-1 tumor cells together with almost all of the other tumor cells, including NK-resistant cells. Nonadherent PEC contained at least two subpopulations of killer cells. One, directed to syngeneic BAMC-1 cells, was both Thy1.2 and asialo GM1 positive, and another, directed to allogeneic YAC-1 cells, was asialo GM1 positive but Thy1.2 negative. A cold target inhibition assay also suggested the presence of more than two subpopulations. These results indicate that T cells play a determined role in the immunotherapeutic effect of OK-432 on BALB/c mice bearing BAMC-1 tumor, although the participation of activated macrophages could not be excluded. The cells responsible for killing BAMC-1 and other tumor cells appearing in the PEC on day 12 were characterized as containing at least two kinds of lymphokine-activated killer cells.

PRODUCTION OF PYROGEN BY POLYMORPHONUCLEAR LEUKOCYTES DURING THE COURSE OF CASEIN‐INDUCED PERITONITIS IN RABBITS

Rabbits with casein-induced peritonitis were febrile only during the early stage (1.5 to 12 hours) of inflammation. At that stage, peritoneal exudate cells (PEC) had preformed endogenous pyrogen (EP), while blood leukocytes and leukocytes obtained at the later stage of the inflammatory process did not. Early PEC consisted of 99% polymorphonuclear leukocytes (PMN). As early PEC and highly purified PMN (almost 100% pure) released almost identical amounts of EP, we concluded that PMN were the EP-producing cells in the early PEC. EP from PMN of rabbits was composed of three factors with similar molecular weight (8,000 to 18,000) but different pI values (pI 7.2, 5.4, and 4.5). EP in the three fractions could not be separated from thymocyte comitogenic factor (TMF) in terms of m.w. and pI values. About 60% of EP activity and 92% of TMF activity in the culture supernatant were present in the pI 7.2 fraction. The pI 7.2 factor was weaker in EP activity and stronger in TMF activity than the other acidic fractions (pI 5.4 and 4.5). After high purification, 42 ng of the pI 7.2 factor had one unit of EP activity: one unit of TMF activity was present in 126 pg of highly purified pI 7.2 factor.

Anti-tumour effect of humoral and cellular immunities mediated by a bacterial immunopotentiator, Lactobacillus casei, in mice.

Administration of a mixture containing Lactobacillus casei YIT 9018 (LC9018) and methylcholanthrene-induced fibrosarcoma (Meth A) cells into the peritoneum of syngeneic BALB/c mice suppressed the tumour growth and protected the mice from tumour death. With the appearance of the anti-tumour activity, serum complement-dependent tumour cytotoxic (CDC) antibody was induced on the 5th day after the administration as a result of the adjuvant effect. The cytotoxic antibody was not found in serum on the 5th day after inoculation of Meth A cells alone, but it was induced before the mice died of the tumours. Adjuvant induction of the cytotoxic serum antibody at an early time was also observed using Kirsten murine sarcoma virus-transformed tumour (K234) cells. Both of these cytotoxic antibodies in sera from Meth A-suppressed and the tumour-bearing mice were specific for the tumour cells and were IgM class, since they were absorbed with rabbit anti-mouse IgM antibody. However, the cytotoxic antibody was not found in the peritoneal cavity which was the tumour inoculation site, but binding antibody against the tumour cells was faintly detected in the region using an enzyme-linked immunoabsorbent assay (ELISA). In neutralization tests, the cytotoxic antibody did not exert anti-tumour activity in recipient mice when it was administered to the mice along with the tumour cells or when it was administered i.v. at the time of tumour inoculation. Moreover, the cytotoxic antibody was not available for the antibody-dependent cell-mediated cytotoxicity (ADCC). These results suggest that the cytotoxic antibody did not exert anti-tumour activity in the tumour-suppressed mice. In contrast, peritoneal exudate cells (PEC) on the 5th day, and PEC and spleen cells on the 15th day after i.p. administration of the mixture exerted strong anti-tumour activity as measured by the Winn test. In conclusion, the adjuvant effect of LC9018 induced tumour-specific humoral and cellular immunities but the anti-tumour activity was dependent only on the cellular effectors of the host. The possible use of LC9018 in tumour immunotherapy is discussed.

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.

In vitro detection of cell-mediated immunity to individual tumor-specific antigens of chemically induced BALB/c fibrosarcomas.

In this study we have analyzed the in vitro cell-mediated cytotoxicity of immune peritoneal exudate cells (PEC), elicited in syngeneic mice against the MCA-induced, TSTA-bearing BALB/c fibrosarcoma CA-2, GI-17 and C-3. The 4 h 51Cr-release assay showed the immune PEC effectors to be specifically cytotoxic to fibrosarcoma used for the immunization, but not to other syngeneic MCA-induced tumors or normal fibroblasts. Cold target inhibition experiments on CA-2 cells confirmed the specificity of the reaction. When PEC, lymph-node and spleen cells from BALB/c anti-CA-2 mice were compared for anti-tumor activity, only PEC were found to kill tumor cells significantly. PEC effectors did not have a significant level of NK or NC activities since they were unable to destroy YAC-1 or WEHI-164 tumor cells. PEC anti-CA-2 were analyzed for the expression of T-cell markers by anti-Thy 1.2, anti-Ly 1.2 and Ly 2.2 monoclonal antibodies. Anti-tumor specific effector cells were identified as mature T cells since they were not adherent to plastic and showed Thy 1.2+, Ly 1.2- and Ly 2.2+ phenotypes. In addition, anti-H-2Kd but not anti-H-2Dd alloantiserum added to target cells, blocked CA-2 tumor lysis, thus supporting the conclusion that the T-cell response against TSTA is H-2 restricted.

The glomerular polyanion (GPA) of the rat kidney. I. Concanavalin-A activated cells affect the glomerular polyanion in vitro.

Rat peritoneal exudate cells (PEC) or human peripheral blood cells stimulated with mitogens in vitro were cultured in tissue culture chambers mounted on rat or human cryostat kidney sections. After 20 h, the cultures were discontinued and the glomerular polyanion (GPA) of the glomeruli was studied using the colloidal iron stain. The results show that in contrast to PHA-stimulated cells, rat PEC or human blood cells stimulated with Con-A are able to reduce GPA stainability. It was further shown that rat PEC activated with Con-A for 20 or 48 h were able to suppress in vitro lymphocyte transformation of syngeneic blood lymphocytes in a co-culture system following mitogenic stimulation. In view of recent concepts according to which disturbance of T cell function is associated with in vivo loss of GPA in some forms of the nephrotic state, we conclude that investigation into the possible in vivo significance of the present observations is worthwhile.

Functional heterogeneity among peritoneal macrophages: III. No evidence for the role of arginase in the inhibition of tumor cell growth by supernatants from macrophages or macrophage subpopulation cultures

The adherent population of peritoneal exudate cells (PE) obtained from rats and mice was analyzed for arginase activity in order to determine whether this enzyme has a role in tumor-growth-inhibitory activity. Freshly obtained tumor-growth-inhibitory rat PE cells had little or no arginase activity compared to the high levels of enzyme activity of mouse PE cells. Even after culturing, rat PE cells contained arginase activity 10 times less than that observed with comparable numbers of cultured or noncultured mouse cells. Subpopulations of mouse and rat PE macrophages, analyzed for arginase activity, showed that the light-density populations from cultured rat PE cells and noncultured mouse PE cells expressed arginase activities greater than that seen with heavy-density cells. However, the light-density rat PE cells expressed significantly less arginase activity than did the mouse cells. In attempts to test whether the inability of tumor cells to grow in supernatants or dialyzed supernatants from PE macrophage cultures is due to an arginine depletion, 200 μg/ml of the amino acid was added to the supernatants. The tumor-growth-inhibitory activities of such supernatants, as well as those from supernatants from highly active light-density rat PE macrophage cultures, were not abrogated by the addition of arginine. There was no correlation between the high levels of arginase activity of light-density PE macrophages and their antitumor activity and no evidence that the tumor-growth-inhibitory activity of rat or mouse PE macrophages in the macrophage-tumor models we studied was due to an arginine depletion.

Characterization of Ia-positive peritoneal exudate cells which augment concanavalin a response of T cells

Ia antigens on mouse peritoneal exudate cells (PEC) as well as splenic non-B cells were detected by radioimmunoassay using various anti-Ia sera and radiolabeled specific anti-allotype sera. To remove B cells from PEC and the spleen cell suspensions, anti-ThB was utilized. This antiserum, in the presence of rabbit complement, effectively abrogated the lipopolysaccharide (LPS) response and depleted more than 95% of Ig-bearing cells of PEC and spleen suspensions. After the treatment with anti-ThB and complement, Ia was still detectable by radioimmunoassay on residual PEC and spleen cells. The subregion specificity of the Ia detected on these non-B cells was found to be I- A and I- E· C. Consistent results were observed in functional assays where the PEC cells which reconstitute the proliferative response of pure T cells to concanavalin A (Con A) were also found to express Ia antigens of the I- A or I- E· C subregion.

Antitumor activity of peritoneal exudate cells induced by cell-wall skeleton of Mycobacterium bovis BCG.

Peritoneal exudate cells (PEC) induced by oil-attached cell-wall skeleton of Mycobacterium bovis BCG (BCG-CWS) in ACI/N rats were tested for their effect on both in vivo and in vitro growth of syngeneic fibrosarcoma cells (AMC-60). Treatment of rats with intraperitoneal injections of BCG-CWS induced regression of syngeneic ascites tumor and increased the number of survivals. Whole PEC and adherent PEC from rats injected intraperitoneally with BCG-CWS inhibited the uptake of tritiated thymidine into the fibrosarcoma cells in an in vitro cytostasis test. This in vitro cytostatic effect was more marked as the ratio of effector to target cells increased. In addition, when tumor cells were inoculated subcutaneously with BCG-CWS activated PEC, tumor takes decreased markedly. Oil-stimulated PEC and normal peritoneal resident cells were inactive in inhibition of tumor growth in vivo and in vitro.

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.

Role of donor immunocompetent cells in allograft rejection.

Allotransplantable lines of the BALB/c (H-2d) Leydig cell tumor C4092 were established in DBA/1 (H-2q) mice after maintenance in organ culture. These modified tumors had reduced immunogenicity but were recognized and rejected by previously sensitized DBA/1 mice. The addition of 3.4 X 10(3)-1 X 10(5) peritoneal exudate cells (PEC) from untreated BALB/c mice to cell suspensions of the modified tumor did not restore Immunogenicity when the cell mixture was Inoculated sc Into DBA/1 recipients. However, when equivalent numbers of PEC or spleen cells were inoculated iv into animals receiving the tumor cell suspension sc, the acceptance of the tumors was significantly reduced or prevented. This indicated that the BALB/c lymphoid cells inoculated iv stimulated the immune system of the host, which in turn recognized the tumor cell inoculum. The lack of effect of the BALB/c lymphoid cells when admixed with the tumor did not support the assumption that the loss of passenger lymphoid cells was responsible for the allotransplantability of grafts after organ culture explantation. Additional evidence was presented that supports our view that the reduced immunogenicity was associated with a phenotypic modification of the tumor cells per se.