Induction of LAK-like cells in the peritoneal cavity of mice by inactivated Candida albicans

We have investigated the effect of multiple administrations of inactivated Candida albicans (CA) cells on induction of non-MHC-restricted antitumor cytotoxic responses both in normal and congenitally athymic (nude) mice. Intraperitoneal inoculation of CD2F1 mice with five doses of 2 x 10(7) CA cells over a 2-week interval was associated with the induction of peritoneal exudate cells (PEC) that mediated natural killer cell activity. These cells, in contrast to those elicited by a single dose of CA, killed both NK-sensitive and NK-resistant tumor target cells in vitro. This broad-spectrum, antitumor cytotoxicity peaked 1 day after the last injection of CA, and decreased to control values within 6 (NK-resistant) or 14 (NK-sensitive target cells) days. Cytotoxicity could be recalled to a high level by a boosting injection of CA or a major mannoprotein-soluble antigen (MP) from the Candida cell wall, given 30 days after multiple CA treatment. Upon a 24-hr in vitro incubation, CA-induced peritoneal immunoeffectors lost their killing activity unless human recombinant interleukin-2 (rIL-2) was added to cultures. The non-MHC-restricted cytotoxic PEC activity induced by CA was mainly associated with nonadherent, nonphagocytic large granular lymphocytes (LGL) which exhibited the following phenotypes: (i) asialo GM1+, Lyt 2.2-, and partially Thy 1.2+ (effectors active against NK-sensitive targets) and (ii) asialo GM1+, Lyt 2.2-, and Thy 1.2+ (effectors active against NK-resistant targets). Nude mice also responded to multiple CA inoculations by displaying high cytotoxic activity against NK-sensitive targets and significant cytotoxicity against NK-resistant targets. This cytotoxicity could be recalled on Day +30, and the cytotoxic effectors involved were highly sensitive to anti-asialo GM1 plus complement treatment. Overall, the results add further experimental evidence to the wide range of immunomodulatory properties possessed by C. albicans, and demonstrate that the majority of antitumor cytotoxic activity induced by fungal cells was due to lymphokine-activated killer (LAK)-like effectors.     

Cytotoxicity of lymphoid cells induced by Maclura pomifera (MP) lectin.

The cytotoxic activity of lymphoid cells stimulated with Maclura pomifera (MP) lectin was investigated. Spleen cells of Lewis (LEW) or Brown Norway (BN) rats induced a cell-dependent release of ⁵¹Cr from syngeneic, allogeneic, and xenogeneic erythrocytes when incubated with MP for 4–16 hr. The activity of MP differed from that of concanavalin A (Con A). MP exhibited a greater activity with spleen cells while Con A was more active when bone marrow cells were tested. Activity induced by MP required the presence of the lectin for at least 4 hr and was inhibited by melibiose, an inhibitor of MP binding. MP also stimulated phagocytosis by peritoneal macrophages of LEW rats, but phagocytosis was not responsible for the cytotoxic effect measured by ⁵¹Cr release. The ability of aggressor cells to bind MP did not correlate with their cytotoxic activity. The cytotoxic activity of spleen cells from athymic nude mice was equivalent to that of cells from euthymic littermates when stimulated with MP.     

Activation of maternal killer cells in the pregnant uterus with chronic indomethacin therapy, IL-2 therapy, or a combination therapy is associated with embryonic demise

We have previously shown that NK lineage cells migrate to the murine decidua of pregnancy; but with advancing gestation, they are progressively inactivated in situ by prostaglandins of the E series (PGE2) secreted by decidual cells and decidual macrophages. We have also shown that the same mechanism inactivates all killer lineage cells in the human decidua, and that this inactivation is at least in part due to a down-regulation of IL-2 receptors and an inhibition of IL-2 production in situ. We examined whether chronic indomethacin therapy (to block prostaglandin synthesis), or a systemic administration of a high dose of IL-2, or a combination of both agents administered to pregnant mice could activate killer cells in situ and interfere with the progress of pregnancy; and if so, whether there was a causal relationship between the two events. Pregnant CD1 mice (Day 5 of gestation) were subjected to chronic indomethacin therapy (14 micrograms/ml in drinking water up to Day 15, or 50 micrograms twice daily sc or ip up to Day 10), high dose IL-2 therapy (25,000 Cetus U of human recombinant IL-2, ip every 8 or 12 hr for 3-5 days), or a combination of the two. These treatments led to pregnancy loss in 89-100% of mice, in contrast to 1% loss in control, vehicle-treated mice. Uterine mononuclear cells isolated from the embryo resorption sites exhibited high killer activity against YAC-1 lymphoma as well as murine trophoblast targets, with NK-like phenotype (Asialo GM-1+, Thy-1-) after indomethacin therapy and LAK-like phenotype (AGM-1+, Thy-1+) after IL-2 or indomethacin + IL-2 therapy. That AGM-1+ killer cells resulted in the pregnancy loss was suggested by the findings that in two of three separate experiments, iv injections of AGM-1 ab into pregnant indomethacin + IL-2-treated mice nearly completely prevented the fetoplacental demise (reducing it to 7.7% from 100%). These results reveal that PGE2-mediated inactivation of killer lineage cells in the decidua in situ is conducive to the survival of the conceptus.     

Lymphocytes infiltrating human ovarian tumors. I. Role of Leu-19 (NKH1)-positive recombinant IL-2-activated cultures of lymphocytes infiltrating human ovarian tumors

Tumor-infiltrating lymphocytes (TIL) were obtained from human ovarian tumors, expanded in the presence of IL-2 in culture and studied for cytotoxicity against fresh autologous and allogeneic ovarian carcinoma (CA) targets. TIL from ovarian tumors grew well in long term cultures, achieving from 8- to 682-fold expansion. TIL cultured with IL-2 were cytotoxic against both autologous and allogeneic fresh ovarian CA targets, and no specificity for autologous tumor could be demonstrated in any of the cultures. In all fresh TIL preparations, CD3+ lymphocytes were the major cell type and contained a high proportion (up to 51%) of activated (IL-2R+) cells as determined by two-color flow cytometry. Sorting of bulk TIL cultures followed by cytotoxicity assays identified the Leu-19+ cells, both CD3+ and CD3-, as effectors of cytotoxicity against autologous and allogeneic tumor cell targets. Cold target inhibition assays showed that allogeneic targets (both ovarian CA and a sarcoma) competed effectively with autologous ovarian CA targets for Leu-19+ effectors in TIL cultures. mAb to Leu-19 or Leu-2a did not block lysis of autologous targets by sorted effectors. OKT3 antibody augmented lysis of autologous targets by CD3+Leu-19- effectors only. These results show that non-MHC-restricted Leu-19+ effectors in cultures of TIL with 1000 U/ml of rIL-2 mediate lysis of autologous and allogeneic tumor cells. The CD3+Leu-19- cells, the main population in these cultures, do not mediate tumor lysis. To determine the phenotype of antitumor effectors in IL-2 cultures of TIL, cell sorting followed by functional assays are necessary.     

The participation of nitric oxide in peritoneal exudate cell cytotoxicity of mice by Fusobacterium nucleatum

Previously we reported that mice infected recurrently with live Fusobacterium nucleatum(Fn) synthesize a significant amount of NO between 12 hr and 24 hr after Fn injection. Fn is a gram-negative rod periodontal pathogen. NO could not be induced by heat-killed Fn or in untreated mice. This NO, derived from the iNOS after infection of live Fn, was not involved in the Fn reduction because Fn clearance occurs within 6 hr. We investigated in this study whether this NO was involved in cytotoxicity in peritoneal exudate cells (PEC) in vivo. The mice were divided into two groups: those treated with live Fn (immune) and those left untreated (normal). PEC number, NO production, detection of apoptosis or death cells, and lactate dehydrogenase (LDH) release activity after injection of live Fn were compared in these groups. In the immune group, the increase of the total cell numbers caused by an increase in neutrophils, a significant NO production only after injection of live Fn at 24 hr and identification of iNOS positive macrophages were confirmed. The apoptotic rate was very low and did not increase at 24 hr in vivo. Therefore, apoptosis was seldom relevant to the NO. In the immune group, LDH activity was remarkable high at 24 hr, and dead cells and macrophages phagocytizing cell fragments increased at the same time. Pretreatment of L NMMA, an inhibitor of iNOS, suppressed LDH activity and cell death. Therefore, the NO derived from the iNOS is involved in the cytotoxicity. These results suggest that NO may contribute to the inflammatory response during Fn infection in periodontitis.     

Successful immunotherapy with intraperitoneal Corynebacterium parvum in a murine ovarian cancer model is associated with the recruitment of tumor-lytic neutrophils into the peritoneal cavity

The rejection of a murine ovarian teratocarcinoma (MOT) after i.p. injection of Corynebacterium parvum was investigated. Treatment with C. parvum (1400 micrograms) 24 hr after i.p. inoculation of a lethal number of tumor cells (10(5] induced an antitumor effect that cured 75 to 95% of the mice. Morphologic analysis and an in vivo cytotoxicity assay that measured the rate of disappearance of radioactivity from the peritoneal cavity after injection of 125IUdR-labeled tumor cells indicated that the antitumor effect was initiated during the first 24 hr after C. parvum injection. During this period of time, host effector cells retrieved from the peritoneal cavity prevented tumor growth in a Winn assay and lysed radiolabeled MOT targets in a 4-hr Cr-release assay. After separation of peritoneal inflammatory cells on a Percoll gradient, neutrophil-enriched fractions demonstrated significant in vitro tumor lysis, but neutrophil-depleted populations were ineffective. Microscopic analysis of lysis at the single cell level confirmed that neutrophils were binding to and lysing MOT targets. Further characterization of these tumor cytolytic neutrophils revealed that they are nylon wool-adherent, not generated in indomethacin-pretreated mice (but effectively generated in whole body-irradiated mice), and achieve lysis within 30 min after binding to MOT targets. These results indicate that neutrophils must be considered potential antitumor effectors that can be recruited by treatment with biologic response modifiers.     

Idiotype vaccination against murine B cell lymphoma. Humoral and cellular requirements for the full expression of antitumor immunity

The roles of humoral and cellular antitumor immune responses induced by immunization with tumor-derived idiotypic IgM were studied in a syngeneic, transplantable B cell lymphoma (38C13) of C3H mice. Id vaccination with keyhole limpet hemocyanin-conjugated Id induced protection against a subsequent lethal tumor challenge. Such immunizations elicited anti-idiotypic antibodies that were cytotoxic in in vitro antibody-dependent cellular cytotoxicity assays as well as in vivo passive transfer experiments. L3T4+ T cells, which proliferated in vitro in response to the specific Id protein, were also induced. However, cells mediating direct cytotoxicity, either in vitro or in vivo, were not observed in the lymph nodes, spleens, or peritoneal cavity of immune mice or at the site of tumor regression as demonstrated by using a tumor sponge implantation model. In addition, in vitro sensitization of immune lymphocytes against 38C13 tumor cells failed to induce cytotoxicity. Immunization with lipid conjugated Id also elicited a T cell proliferative response but failed to induce anti-idiotypic antibodies and did not confer resistance to tumor growth. These results suggest that anti-idiotypic antibodies play the major role in the destruction of 38C13 tumor cells. However, in vivo depletion of L3T4+ or Lyt-2+ cells from 38C-Id-keyhole limpet hemocyanin-immunized mice resulted in diminished protection against a tumor challenge. Thus, although humoral responses appear to play the predominant part in tumor destruction, cellular responses are also required for the full expression of antitumor immunity in this system.     

The capacity of activated murine macrophages for augmented binding of neoplastic cells: analysis of induction by lymphokine containing MAF and kinetics of the reaction

The capacity for augmented binding of tumor cells is an initial and necessary part of macrophage-mediated tumor cytotoxicity. To study the induction of binding capacity, we obtained FCS-elicited, inflammatory macrophages from C57BL/6J mice. Exposure of these macrophages to lymphokine(s) containing MAF induced augmented binding capacity in a dose-dependent fashion. Resident peritoneal macrophages did not respond to lymphokine, and endotoxin did not appreciably influence induction of binding. Maximum induction of binding required continuous interaction between macrophages and lymphokine for 6 to 10 hr. The conditions necessary for induction of binding closely paralleled those for induction of priming or cytolysis. Exposure of FCS-elicited macrophages from C3H/HeJ mice, although not of macrophages from A/J mice, induced augmented binding. The data suggest that the augmented capacity for binding tumor cells is induced by lymphokine(s) and that a major part of induction of priming for cytolysis by MAF is induction of such binding.     

Rotenone-induced caspase 9/3-independent and -dependent cell death in undifferentiated and differentiated human neural stem cells

We used human neural stem cells (hNSCs) and their differentiated cultures as a model system to evaluate the mechanism(s) involved in rotenone (RO)- and camptothecin (CA)-induced cytotoxicity. Results from ultrastructural damage and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining indicated that RO-induced cytotoxicity resembled CA-induced apoptosis more than H(2)O(2)-induced necrosis. However, unlike CA-induced, caspase 9/3-dependent apoptosis, there was no increased activity in caspase 9, caspase 3 or poly (ADP-ribose) polymerase (PARP) cleavage in RO-induced cytotoxicity, in spite of time-dependent release of cytochrome c and apoptosis-inducing factor (AIF) following mitochondrial membrane depolarization and a significant increase in reactive oxygen species generation. Equal doses of RO and CA used in hNSCs induced caspase 9/3-dependent apoptosis in differentiated cultures. Time-dependent ATP depletion occurred earlier and to a greater extent in RO-treated hNSCs than in CA-treated hNSCs, or differentiated cultures treated with RO or CA. In conclusion, these results represent a unique ultrastructural and molecular characterization of RO- and CA-induced cytotoxicity in hNSCs and their differentiated cultures. Intracellular ATP levels may play an important role in determining whether neural progenitors or their differentiated cells follow a caspase 9/3-dependent or -independent pathway in response to acute insults from neuronal toxicants.     

Effect of in vivo administration of different adjuvants on the in vitro candidacidal activity of mouse peritoneal cells

The candidacidal activity (CA) of peritoneal cells (PC) in vitro was used as a measure of nonspecific microbicidal activity of phagocytes after intraperitoneal injection of mice with different adjuvants. Dilutions of PC were incubated with constant numbers of C. parapsilosis in a 96-well culture plate. The PC number causing 50% reduction of yeast colonies formed after 48 hr at 37 degrees C was called 1 CA50 unit. CA was expressed in CA50 units per 10(6) PC. Optimal reduction of the number of viable candida cells in vitro was established within 1.5 hr while 50% reduction was reached after 0.5 hr. In this test CA was, within limits, independent of the number of viable candida cells added per well (22 to 152 yeast cells), of the concentration of fetal calf serum (1-20%) and of the presence of heat-labile serum components. The CA of PC of individual mice was measured 6, 24, and 96 hr after injection of an adjuvant. In most instances optimal CA was observed 6 hr after administration of adjuvant and varied from 3.7 (methylamine) to 50 (Corynebacterium parvum strain 4982) units. With respect to the titer and duration of CA, the adjuvants were arranged in the following order of increasing efficacy: methylamine, heparin, polyol L 121, suramin, dextran sulfate, polyol L 101, dimethyldioctadecylammonium bromide, Liquoid, heat-killed Listeria monocytogenes, formalin-killed C. parvum strain 10387, and strain 4982. The CA induced by the latter strain persisted at least till 96 hr after injection. The induction of CA was accompanied by recruitment of polymorphonuclear cells. The contribution of distinct phagocytic effector cells to CA and the correlation between modulation of the specific and nonspecific immunity are discussed.     

Mechanisms of in vivo generation of cytotoxic activity against allograft: 1. Local differentiation of mature cytotoxic T lymphocytes in rejection of allogeneic lymphocytes

Although cytotoxic activity was not detected within the spleen and regional lymph nodes from mice immunized sc with allogeneic lymphocytes, such activity was detected consistently in glass-nonadherent and anti-θ-sensitive peritoneal exudate cells (PE cells) from Day 5 after immunization and reached a maximum by Day 7. Immunized spleen cells developed cytotoxic T lymphocytes (CTLs) earlier and more effectively than normal spleen cells when transferred ip into X-irradiated syngeneic normal mice together with immunizing antigen, while they did not become cytotoxic when transferred without antigen. These results suggest that spleen and lymph node cells which may have differentiated into some transitional state by in vivo immunization may differentiate into mature CTLs, following direct contact with antigen at the site of graft. CTLs generated there appear to be responsible for the rejection of allogeneic lymphocytes. Cytotoxicity of PE cells was also generated in X-irradiated mice and augmented cytotoxicity was generated by treatment with cyclophosphamide.     

Nonspecific stimulation of host defense by Corynebacterium kutscheri. I. Antitumor effect

The effect of local injection of formalin-killed Corynebacterium kutscheri (FK.CK) on mouse survival after the intraperitoneal inoculation of Ehrlich ascites carcinoma in outbred ddY mice or P388 leukemia cells in inbred CDF1 mice was investigated. Treatment of mice in the dose range of greater than 10(6) organisms per mouse conferred the substantial protection on both mice. The initial phase of antitumor effect consisted of the marked increase in the number of peritoneal exudate cells and the enhanced cytotoxicity of peritoneal exudate cells. The Winn assay disclosed that antitumor effect by which tumor-burden mice could survive was attributable to nonadherent splenocytes whose activity was impaired by treatment with anti-T cell serum and complement. A single injection of FK.CK induced the cytotoxicity to three different murine tumor cells in serum of treated mice without a boosting injection of endotoxin. Furthermore, the generation of effector cells and serum cytotoxicity seemed to be paralleled by that of the delayed-type hypersensitivity to this organism. Thus, the antitumor resistance induced by C. kutscheri is considered to be in part T cell mediated.     

Multifunctional CD4 cells expressing gamma interferon and perforin mediate protection against lethal influenza virus infection

CD4 effectors generated in vitro can promote survival against a highly pathogenic influenza virus via an antibody-independent mechanism involving class II-restricted, perforin-mediated cytotoxicity. However, it is not known whether CD4 cells activated during influenza virus infection can acquire cytolytic activity that contributes to protection against lethal challenge. CD4 cells isolated from the lungs of infected mice were able to confer protection against a lethal dose of H1N1 influenza virus A/Puerto Rico 8/34 (PR8). Infection of BALB/c mice with PR8 induced a multifunctional CD4 population with proliferative capacity and ability to secrete interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF-α) in the draining lymph node (DLN) and gamma interferon (IFN-γ) and IL-10 in the lung. IFN-γ-deficient CD4 cells produced larger amounts of IL-17 and similar levels of TNF-α, IL-10, and IL-2 compared to wild-type (WT) CD4 cells. Both WT and IFN-γ(-/-) CD4 cells exhibit influenza virus-specific cytotoxicity; however, IFN-γ-deficient CD4 cells did not promote recovery after lethal infection as effectively as WT CD4 cells. PR8 infection induced a population of cytolytic CD4 effectors that resided in the lung but not the DLN. These cells expressed granzyme B (GrB) and required perforin to lyse peptide-pulsed targets. Lethally infected mice given influenza virus-specific CD4 cells deficient in perforin showed greater weight loss and a slower time to recovery than mice given WT influenza virus-specific CD4 cells. Taken together, these data strengthen the concept that CD4 T cell effectors are broadly multifunctional with direct roles in promoting protection against lethal influenza virus infection.     

The effects of neuraminidase and galactose oxidase on murine lymphocytes. I. Evidence for the differential delivery of signal(s) leading to cell proliferation and the differentiation of cytotoxic T cells.

The sequential treatment of normal C57BL/6 mouse spleen cell populations with neuraminidase (NA) and galactose oxidase (GO) resulted in cell proliferation, but not in the differentiation of cytotoxic T cells. In contrast, C57BL/6 spleen cells derived from animals primed 5 to 8 months earlier with alloantigen (P815 mastocytoma cells of the DBA/2 strain) both proliferated and demonstrated T cell-mediated cytotoxicity after NAGO stimulation. T cells differentiating into cytotoxic cells after NAGO treatment demonstrated properties similar to alloantigen-specific 'memory' T cells. These were: 1) cytotoxicity developed only from 'primed' cell populations, 2) cytotoxicity developed within 24 hr after NAGO treatment, 3) DNA synthesis was not required for the differentiation of cytotoxic cells during the first 24 hr of culture but both DNA synthesis and cell proliferation were required for the cytotoxicity developing after 24 hr, and 4) all cytotoxicity induced by NAGO showed specificity for the priming alloantigen. It was found, furthermore, that cytotoxicity could be induced at much lower GO concentrations than needed for increased DNA synthesis. We interpret this finding as an indication that NAGO can differentially deliver two 'signals' to T lymphocytes: one leading to cell proliferation, the other causing the differentiation of memory T cells into cytotoxic effectors.     

Appearance of a Cell Surface Antigen Associated with the Activation of Peritoneal Macrophages in Mice

A relatively large population of murine peritoneal exudate macrophages induced with viable BCG or heat-killed Corynebacterium parvum was stained by the antiserum prepared against purified gangliotetraosyl ceramide (asialo GM1), while only a small population of peritoneal resident macrophages or peritoneal exudate macrophages induced with proteose peptone was stained. The cytotoxicity assay of those macrophages with anti-asialo GM1 plus complement supported these results. Peritoneal macrophages induced with BCG or C. parvum showed strong cytotoxicity for EL4 cells in vitro, while resident or peptone-induced peritoneal macrophages showed no cytotoxicity. BCG- or C. parvum-induced peritoneal cells contained both NK cells and cytotoxic macrophages, and either in vivo or in vitro pretreatment of the cells with anti-asialo GM1 and complement abolished the activities of both types of cells. Peptone-induced peritoneal macrophages incubated with lymphokines (LK) or lipopolysaccharide (LPS) were cytotoxic for EL4 cells and contained an increased number of cells stained by anti-asialo GM1. The cytotoxicity of these in vitro activated macrophages was reduced by treatment with anti-asialo GM1 plus complement. When peptone-induced peritoneal macrophages were incubated with LK, the number of cells stained by anti-Ia antiserum increased, but the number did not increase when the macrophages were incubated with LPS. Pretreatment of peptone-induced macrophages with anti-asialo GM1 plus complement did not affect the ability of the macrophages to be activated by LK. These results taken together strongly suggest that the antigen (s) reactive with anti-asialo GM1 is expressed on the cell surface of cytotoxic peritoneal macrophages in mice.     

The relative contribution of resident pulmonary alveolar macrophage and inflammatory polymorphonuclear neutrophils in host resistance to pulmonary infection by Candida albicans

Cortisone (CA) or cyclophosphamide (Cy) treatment of mice was used to investigate the relative contributions of pulmonary alveolar macrophages (PAM) and inflammatory neutrophils (PMN) in the initial defense against intratracheal challenge (IT) with Candida albicans. Mice treated with either CA or Cy were susceptible to IT challenge with 10–100 x less C. albicans than were untreated mice. Untreated mice rapidly eliminated C. albicans from their lungs with the majority of the organisms being cleared within three hours of challenge. Mice treated with CA initially cleared some of the C. albicans but were unable to clear all the C. albicans as did the untreated mice. Mice treated with Cy were unable to clear C. albicans from their lungs. Candida albicans did not disseminate from the lungs of untreated mice, while in both of the treated groups, C. albicans disseminated to the liver, spleen, brain and kidneys, rapidly killing the treated hosts. Analysis of the changes in cells in lung lavage fluids collected at various times after C. albicans challenge, revealed that large numbers of PMN accumulated in the lungs of both untreated and CA-treated mice, whereas PMN were virtually undetectable in lavage fluids from Cy-treated mice. Resident PAM from untreated mice were able to kill approximately 70 % of 10⁵ C. albicans in a 3 hr in vitro killing assay. By contrast, at similar effector: target ratios, resident PAM from Cy-treated mice killed only about 20% of the inoculum and resident PAM from CA-treated mice were unable to kill C. albicans. PMNs from both untreated and CA-treated mice killed approximately 70% of 10⁵ C. albicans in vitro. The data indicates that both PAM and PMN were critical to the initial clearance of C. albicans from pulmonary tissue. The accumulation of PMN in the lungs appeared to be required for the complete clearance of C. albicans from the lungs yet was not sufficient to inhibit dissemination of C. albicans from the lungs in CA-treated mice. The presence of PAM with in vitro candidacidal abilities appeared to be required for both the clearance of C. albicans and inhibition of dissemination of C. albicans from the lungs. Compromise of either PAM or PMN function can lead to increased pulmonary susceptibility to C. albicans.     

Macrophage activation for tumor cytotoxicity: induction of tumoricidal macrophages by supernatants of PPD-stimulated Bacillus Calmette-Guérin-immune spleen cell cultures.

Resident peritoneal macrophages from normal mice were activated for tumor cytotoxicity in vitro by co-cultivation with BCG1-immune spleen cells and PPD and by incubation with supernatants of PPD-stimulated BCG-immune spleen cell cultures (lymphokine supernatants). Lymphokine activation of macrophages occurred in unfractionated PC suspensions as well as in macrophage monolayers depleted of nonadherent PC. Tumor cytotoxicity by lymphokine-activated macrophages was evident by 3 to 4 hr of culture in active supernatants, reached maximal levels by 8 to 12 hr. and was absent by 20 hr. Continued incubation in lymphokines or even re-exposure after washing did not maintain macrophage cytotoxicity. The capacity of normal resident macrophages to be activated by lymphokines in vitro progressively decreased and was absent by 20 hr in culture. This decrease did not necessarily reflect cell death; macrophage viability as estimated by exclusion of trypan blue or by phagocytic responses did not change over the 20-hr culture period. The short lived nature of both macrophage tumoricidal capacity and capacity of precursor cells to be activated by lymphokines may function as negative feedback mechanisms in immune reactions.     

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.     

Trafficking and activation of murine natural killer cells: differing roles for IFN-gamma and IL-2

The repeated ip injection of highly purified recombinant IFN-gamma or IL-2 resulted in a local increase in peritoneal NK activity. This increase in lytic activity was paralleled by increases in the number of peritoneal leukocytes reacting with a rat monoclonal antibody directed against the NK cell-associated surface antigen LGL-1. LGL-1 reacts specifically with the majority of murine NK cells in BALB/c and C57BL/6 mice. A single injection of IFN-gamma induced more peritoneal NK activity at 24 hr than IL-2 on a protein basis. Both cytokines induced increases in the number of LGL-1+ peritoneal cells by 24 hr after injection. Simultaneous injection of suboptimal amounts of IFN-gamma (100 U) and IL-2 (10,000 U) resulted in a significant augmentation of peritoneal NK activity over that observed with either cytokine alone. Also, the peritoneal NK activity generated in response to ip injection of high doses of IL-2 (100,000 U) could be dramatically reduced by simultaneous injection of a neutralizing monoclonal antibody to IFN-gamma. Administration of IFN-gamma 1 day prior to IL-2 resulted in a significant augmentation of the NK activity above that observed with the individual cytokines. In contrast, injection of IL-2 prior to IFN-gamma did not enhance NK activity over that observed with the individual cytokines. Both cytokines must be injected ip for the complementary effects of IFN-gamma and IL-2 on peritoneal NK activity to occur. In contrast, in vitro incubation of peritoneal leukocytes with IFN-gamma resulted in neither a significant enhancement of NK lytic activity nor an increase in the number of LGL-1+ cells. In vitro treatment of peritoneal leukocytes with IL-2 always resulted in significant augmentation of NK lytic activity in the absence of any increase in the number of LGL-1+ cells. These data are consistent with the hypothesis that the local release of IFN-gamma increases peritoneal NK activity by promoting the influx of blood-borne LGL-1+ NK cells from other sites. In contrast, low doses of IL-2 augment the lytic activity of local resident NK cells, whereas high doses of this cytokine induce both an activation of local NK cells and emigration of LGL-1+ NK cells from other sites due to the endogenous generation of IFN-gamma within the peritoneal cavity. Therefore, the local release of IFN-gamma may play an important role in regulating NK cell infiltration in vivo.     

Modulation of natural killer activity in mice following infection with Listeria monocytogenes

Mice that received a sublethal, intraperitoneal dose of viable Listeria monocytogenes, virulent strain 10403, exhibited a systemic increase in natural killer (NK) activity. The kinetics of the response differed with respect to the various effector cell populations analyzed. Resident peritoneal cells and peripheral blood leukocytes demonstrated high NK activity on Days 3, 7, and 10. Peak spleen and bone marrow NK activity was observed on Day 3, returning to normal levels by Day 7. In contrast, peritoneal exudate cells, elicited with proteose peptone, expressed enhanced NK activity for 60 days following infection with viable Listeria. Augmented NK activity was detected with all cell types as early as 12 hr after infection. The intraperitoneal injection of nonviable antigenic preparations derived from L. monocytogenes, strain 10403, resulted in the enhancement of peritoneal and splenic NK activity. In contrast, mice that received an intraperitoneal injection of avirulent Listeria, strain 19113, failed to express enhanced levels of NK activity. The genetic trait of anti-listerial resistance which is associated with non-H-2 linked genes was of no importance with respect to enhanced NK activity. Listeria-resistant C57BL/6J and Listeria-susceptible DBA/2J mice both produced systemic augmentation of NK activity following infection. NK activity was not abrogated by macrophage depletion or by treatment with anti-Thy 1.2 serum plus complement. These results confirm the potent immunostimulatory capacity of virulent Listeria for NK activity and provide further insight into the kinetics of this response in various lymphoid compartments. The protracted augmentation of NK activity of elicited peritoneal exudate cells as compared to nonelicited peritoneal cells in Listeria-primed mice suggests that the influx of inflammatory cells may provide NK-enriched and/or accessory populations for immunopotentiation of NK activity in inflammatory sites.