Immune Peritoneal Exudate Research Articles

Responses of mouse lymphocytes to extracellular adenosine 5'-triphosphate (ATP). Lymphocytes with cytotoxic activity are resistant to the permeabilizing effects of ATP.

The effects of extracellular ATP on plasma membrane permeability in mouse lymphocytes were studied with plasma membrane depolarization, uptake of ethidium bromide, and release of lactate dehydrogenase as indicators of increased permeability. Extracellular ATP induced sustained depolarization of plasma membrane potential as well as uptake of low m.w. fluorescent markers in mouse lymphocytes derived from thymus and spleen, and in two lymphoma lines YAC-1 and MBL-2. The fully ionized form ATP4- rather than MgATP2- mediated the increased permeability of the plasma membrane. Although prolonged exposure to exogenous ATP ultimately lysed the lymphocytes, two CTL populations (CHM-14 clone and CTLL-2 line) and IL-2-treated spleen lymphocytes with unrestricted killing activity were highly resistant to the permeabilizing action of extracellular ATP at all concentrations tested. In addition, CTL derived from primary immune peritoneal exudate and enriched by in vitro culture for 5 days in the presence of specific stimulator cells were also resistant to this permeabilizing effect. These findings show that exogenous ATP has a lytic effect on mouse lymphocytes but not on CTL, and suggest a role for ATP in cell-mediated cytotoxicity.

Effects of Staphylococcus epidermidis on Cellular Immunity to Infection with Listeria monocytogenes

With the present study, the effects of intravenous applications of Staphylococcus epidermidis (SE) on the course of experimental infections of mice with Listeria monocytogenes were evaluated. SE treatment 24 h prior to Listeria infection led to a reduced growth of Listeria organisms in both livers and spleens and to an increased resistance of infected animals against a lethal Listeria challenge. SE treatment 24 h after Listeria infection resulted in an enhanced growth of and retarded elimination of Listeria organisms from animal organs as well as in a reduction of delayed-type hypersensitivity to soluble Listeria antigen. Adoptive immunotherapy accomplished by transferring immune peritoneal exudate T-lymphocyte-enriched cells (PETLEs) to Listeria-infected recipients 24 h before SE treatment did not prevent the delay in clearance of Listeria organisms. When Listeria-infected recipients compromised in their immune response by SE treatment were infused with immune PETLEs either immediately or 24 h after the application of SE, the immunosuppression induced by SE proved to be reversible. It is concluded that, in analogy to other bacterial immunomodulators, Staphylococcus epidermidis is able to either nonspecifically activate macrophages or interfere with T-lymphocyte functions.

Specific tumoricidal activity of cytotoxic macrophages and cytotoxic lymphocytes.

The antitumor potency and specificity of syngeneic immune peritoneal exudate cells were tested. Groups of DBA/2 mice were immunized against syngeneic SL2 tumor cells. Then 6 days after the last immunization the antitumor potency, and the specificity of the immunization reaction was tested by injecting groups of the immunized mice with 10(3) to 5 X 10(7) DBA/2 derived L1210, L5178Y, P815 or SL2 tumor cells, and injecting immune peritoneal exudate cells into DBA/2 mice which had been injected 2 h earlier i.p. with 2 X 10(4) or 2 X 10(5) L1210, L5178Y, P815, or SL2 cells. Furthermore the tumor specific cytotoxicity in vitro of isolated immune (vs SL2) peritoneal macrophages was tested against L1210, L5178Y, P815, and SL2 cells. The "reciprocal" experiments (previous immunization against L1210, L5178Y, or P815 cells and 'challenge' with SL2) were also done. Finally, we tested the tumor-specific cytotoxicity of isolated immune peritoneal T-lymphocytes. It was shown that the rejection of tumor cells in previously immunized mice, the antitumor efficacy of the transferred immune peritoneal exudate cells and the in vitro cytotoxicity of purified immune peritoneal macrophages and lymphocytes, were tumor-specific reactions. That is only between the SL2 and L5178Y tumors were cross-reactions observed. However, this cross-reaction was not found at the level of cytotoxic T-cells. This suggests that cytotoxic T-cells and cytotoxic macrophages probably have different mechanisms of recognition of the specific tumor target cells. Treatment of macrophage monolayers, prepared from macrophages of immunized mice, with monoclonal anti-Thy-1 antibodies plus complement caused no decrease in cytotoxicity. This shows that macrophages can really express specific cytotoxicity. Tumoricidal macrophages probably obtain their tumor specificity through the activities of tumor-specific factors produced by sensitized T-cells.

Distinct T-cell proliferative responses to 13762A rat mammary adenocarcinoma and derived clones

We examined the in vitro responses of immune lymphocytes to the tumor antigens of the syngeneic rat mammary adenocarcinoma 13762A. This tumor readily metastasizes to lymph node and lungs and is poorly immunogenic. Rats were immunized with a highly immunogenic clone (18A) which was isolated as a spontaneous variant from the parental 13762A tumor. Clone 18A grew progressively in irradiated rats but regressed completely in normal rats. Animals immune to 18A tumor were also immune to parental 13762A. Lymphocytes obtained from the spleen and peritoneum of immune rats were tested for specific proliferation to parental 13762A tumor and clone 18A to determine whether similar cross-reactivity to these tumors occurred in vitro. We found an anatomical difference in localization of immune lymphocytes which reacted to the two tumor cell lines. Immune peritoneal exudate cells (PEC) responded strongly to clone 18A but poorly to 13762A, while immune spleen cells from the same animals responded predominantly to 13762A tumor. After 7 days culture, PEC proliferating in response to clone 18A contained 84–95% W3/25 + T-helper cells, and only 5–8% OX8 + cytotoxic/suppressor cells, while analogous cultures of spleen cells responding to parental 13762A tumor consisted of 60–80% W3/25 + cells and 20–23% OX8 + cells. Immune spleen cell cultures stimulated with 13762A tumor generated cytotoxic lymphocytes which specifically lysed both parental 13762A and clone 18A cells. We conclude that despite cross-reactivity in vivo and in vitro, antigens present on 13762A and 18A tumor cells stimulated different subsets of immune T cells.

Cellular cooperation in the expression of murine delayed-type hypersensitivity (DTH): 1. A normal accessory cell population enhances DTH produced by immune peritoneal exudate T lymphocytes

A murine system for local passive transfer of delayed-type hypersensitivity (DTH) has recently been described. It was determined that untreated and T-lymphocyte-enriched (nylon-wool-nonadherent) fractions of peritoneal exudate (PE) cells from immunized donors could be transferred with soluble antigen to normal recipient footpads to efficiently produce a local DTH response. Untreated spleen or lymph node (LN) cell populations were strikingly less capable in this regard. It is now reported that addition of normal untreated PE cell populations to immune T-enriched PE cells markedly enhanced the DTH response transferred by the latter. Specific swelling was dose dependent with respect to each cell type. Removal of T lymphocytes from the normal PE cell population did not affect its enhancement of DTH. By cotransfer of 1 × 10 7 normal PE cells, significant specific swelling was obtained using 1–3 × 10 5 T-enriched immune PE cells. This represented a three- to seven-fold reduction in the requirement for the latter cell type. This scheme of DTH enhancement was employed to evaluate the mechanisms for decreased capability of immune LN and spleen for DTH transfer when compared to PE. No evidence was found that either adherent or nonadherent suppressor cells are operative at the time of DTH expression. Cotransfer of a DTH-enhancing population failed to equalize DTH expression by LN and spleen with that of PE. It is concluded that DTH effector-T-cell activity is enriched in immune peritoneal exudate and that non-T-cell population(s) from that source actively enhance DTH expression.

Amblyomma americanum: Requirement for host Fc receptors in antibody-mediated acquired immune resistance to ticks

Guinea pig recipients of anti-tick immune serum or immune peritoneal exudate cells expressed 25 and 30% tick rejection, respectively, when challenged with Amblyomma americanum larval ticks. Previous studies have shown that IgG 1 antibodies are responsible for the ability of immune serum to transfer resistance to ticks and to mediate the accompanying, and required, cutaneous basophil response. Since IgG 1 antibodies induce mast cell-mediated passive cutaneous anaphylaxis and cutaneous basophil responses by interaction with cell surface Fc receptors, we investigated whether host Fc receptors were involved in the mechanism of antibody-mediated immune resistance to ticks. Recipients of immune serum pretreated intravenously with rabbit IgG failed to express resistance when challenged. In contrast, recipients of immune peritoneal exudate cells similarly pretreated expressed normal resistance. Sheep IgG had no inhibitory effect on the transfer of resistance by either immune serum or peritoneal exudate cells. Furthermore, recipients of immune serum pretreated with the Fc fragment from papain digestion of rabbit IgG failed to express resistance when challenged with ticks. Rabbit Fab and sheep Fc and Fab had no effect on the transfer of resistance by immune serum. Purity of rabbit Fc preparations was verified by the ability to inhibit mast cell-mediated passive cutaneous anaphylaxis due to high-titered IgG 1 antiovalbumin antibodies. Rabbit Fab and sheep Fc fractions did not inhibit passive cutaneous anaphylaxis reactions. These findings suggest that immunoglobulin Fc receptors on host cells, such as mast cells and basophils, are required for antibody-mediated immune rejection of ticks from guinea pigs.

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 existence of antibody-like activities against the weakly immunogenic minor histocompatibility antigens

BALB/c mice develop cytotoxic lymphocytes as well as produce specific antibodies against the minor histocompatibility antigens when injected with DBA/2 P815 cells. P815 cells grown in BALB/c mice have IgG antibodies on their surface as demonstrated by the binding of 125I-labeled goat anti-mouse IgG and by complement-dependent cytotoxicity. Serum from BALB/c mice hyperimmunized with P815 cells blocked lymphocyte-mediated cytotoxicity by BALB/c immune peritoneal exudate cells. This blocking activity was removed by absorbing hyperimmune serum with DBA/2 spleen cells or P815 cells. This result suggests that specific antibodies were generated against the minor histocompatibility differences between BALB/c and DBA/2 mice. The experimental procedures described may be very useful in demonstrating minute quantities of antibody against minor histocompatibility antigens on tumor cells.

Reduction of nitroblue tetrazolium for functional evaluation of activated macrophages in the cell-mediated immune reaction

In phagocytic cells nitroblue tetrazolium (NBT) is reduced to formazan. This, in turn, is deposited as a dark blue precipitate in the cytoplasm. Stimulation of the hexosemonophosphate (HMP) shunt leads to increased reduction of NBT. Changes in reduction capacity can be quantitated by the extraction and photometrical evaluation of formazan. Data presented here demonstrate that quantitative evaluation of intracellular formazan is a sensitive indicator for mediators of cellular immunity (MCI) activating marcophages. The NBT reduction potential was increased by a factor of 3.7 if bovine γ globulin (BGG) immune peritoneal exudate (PE) cells were incubated with BGG or if MCI-rich culture supernatants were transferred to normal PE cells. The increased NBT reduction was always paralleled by increased inhibition of capillary migration and by positive skin testing. These experiments suggest that the test for NBT reduction may be a quantitative evaluation of MCI activity.

Immunity to antigenically related salmonellae: effects of humoral factors on the bactericidal activity of normal and immune peritoneal exudate cells.

Immunity against Salmonella enteritidis and Listeria monocytogenes was studied by measuring in vitro the bactericidal activity of peritoneal exudate cells (PEC) of control (normal PEC) and S. typhi Ty2-immune (immune PEC) mice. Specific immune serum, anti-S. tyhphi Ty2, heat inactivated at 56 degrees C for 30 min, significantly inhibited the growth of S. enteritidis only with immune PEC. These opsonic factors had no effect upon the activity of normal PEC. That such inhibition could not be demonstrated in Listeria experiments, either with immune or normal PEC, suggests that S. enteritidis was specifically recognized, in vitro, by the thermostable opsonin anti-S. typhi Ty2 and that macrophages from immune PEC were more efficient in inhibiting bacterial growth than those from normal PEC. Thus, the interaction between macrophages and the microorganism seems to play an essential role in cell-mediated as well as humoral immunity.

Functional heterogeneity among peritoneal macrophages: I. Effector cell activity of macrophages against syngeneic and xenogeneic tumor cells

Peritoneal exudate cells from immunized and nonimmunized animals were separated into subpopulations by centrifugation on discontinuous bovine serum albumin (BSA) density gradients. Cells in the several subpopulations were then tested for their cytostatic or cytotoxic activity against syngeneic and xenogeneic tumor cells. Nonimmune macrophages isolated at the 8 to 11% BSA interface were highly inhibitory to the growth of syngeneic and xenogeneic tumor cells during coculture for 24 to 48 hr. A second macrophage subpopulation of heavier density was not as effective in preventing tumor growth and frequently augmented it. Cytotoxic activity against (C58NT) D tumor cells could not be detected with macrophages or subpopulations of macrophages from immune as well as nonimmune animals, as determined by a 4-hr chromium release assay. The cytotoxic activity of the immune peritoneal exudate cells observed by this assay could be accounted for by the small percentage of lymphocytes present.

Changes in Fluorescence Polarization of a Membrane Probe during Lymphocyte-Target Cell Interaction

Abstract Interaction of exposed membrane-lipid regions after mixing of immune peritoneal exudate T lymphocytes (PEL) and target cells (TC) was studied by monitoring the degree of fluorescence polarization (P) of 1,6-diphenyl 1,3,5-hexatriene (DPH), a lipophilic probe, which can be transferred upon contact of lipid phases. Since the P of DPH-labeled PEL and labeled TC differed significantly, detection of DPH translocation between labeled and nonlabeled cells was possible. A decrease in P was observed after interaction of DPH-labeled PEL and nonlabeled TC. Conversely, an increase in P was detected after nonlabeled PEL were allowed to react with DPH-labeled TC. These changes in P were evident when TC were reacted with specific PEL. However, substantial changes were also monitored with PEL immunized against third-party target cells. No significant changes in P were obtained when DPH-labeled TC were reacted with normal or immune lymph node cells or thymocytes, or with anti-TC antibody. These findings suggest that nonspecific collision of exposed membrane lipid regions occurs during immune PEL-TC interaction. We propose that binding of cytotoxic lymphocytes and TC is preceded by an unstable and nonspecific hydrophobic interaction of cells, which facilitates specific and stable binding. In support of this proposal is the finding that changes in the composition of membrane lipid constituents prevent binding of PEL and TC.

Cell-Mediated Immunity in Mice Against Papain-Solubilized Histocompatibility and Tumor-Specific Antigens by a Macrophage Migration Inhibition Microassay

The cell-mediated immune status of B10.D2 (H-2d) mice immunized with spleen cells from a congenic strain, B10.A (H-2a), differing at the H-2 locus and of BALB/c mice immunized with a syngeneic simian virus 40 (SV40)-induced sarcoma (mKSA-TU5) was evaluated by an agarose microassay for migration inhibition factor. The inducing antigens in this experiment were papain-solubilized and partially purified chromatographic preparations of spleen cells from A/J mice (H-2a) and a papain-solubilized antigen extract prepared from a tissue culture-adapted cell line (TU-5), derived from the SV40-induced mKSA tumor. The assay used microliters of normal or immune peritoneal exudate cells (PEC) resuspended in a 2-mul droplet of agarose and cultured in the presence or absence of antigen. Specific migration inhibition of PEC from immunized mice was observed with concentrations of solubilized antigen preparations as low as 2.0 mug/ml (3.67 mug/chamber).

Lymphocyte-mediated cytolysis of allogeneic tumor cell in vitro: II. Binding of cytotoxic lymphocytes to formaldehyde-fixed target cells

Allogeneic immune peritoneal exudate lymphocytes would bind to viable monolayers of EL4 target cells but not to EL4 monolayers killed by heat, dehydration, osmotic shock, or other procedures. Fixation with formaldehyde in low concentration killed the EL4 monolayers but did not abolish binding. Formaldehyde-treated EL4 cells also permitted binding to varying degrees after further manipulations which originally abolished binding to unfixed cells.

Antigen-Macrophage Interaction

Abstract Antigen-stimulated tritiated thymidine (3H-TdR) incorporation by lymphocytes is an in vitro correlate of cellular immunity. The stimulation of this response by hapten-carrier conjugates may be inhibited by free hapten, a finding previously interpreted as evidence for independent hapten-specificity of thymus dependent (T) lymphocyte receptors. We confirm the specific inhibition by dinitrophenyl (DNP)-lysine of DNP-guinea pig albumin (GPA) induced stimulation of lymphoid cell cultures from guinea pigs immunized with DNP-GPA and report a similar specific inhibition caused by DNP-ovalbumin (OVA). Nonetheless, our results indicate that this hapten-specific inhibition is not due to blockade of T cell receptors specific for hapten-carrier conjugates. Thus we show: 1) DNP-OVA must be present before and during “pulse” exposure to DNP-GPA but has no effect if added after such a pulse. 2) Preincubation of macrophage-rich peritoneal exudate cells with DNP-OVA blocks the capacity of these cells to bind low concentrations of DNP-GPA and then to stimulate immune peritoneal exudate lymphocytes to divide. 3) Preincubation of DNP-GPA immune peritoneal exudate lymphocytes in DNP-OVA has no effect on their capacity to subsequently respond to macrophage-associated DNP-GPA. We conclude that the capacity of DNP-OVA to inhibit DNP-GPA stimulation of 3H-TdR incorporation by lymphoid cell populations from guinea pigs immune to DNP-GPA is due to competition for a hapten-specific cytophilic antibody on the surface of the macrophage and not to competition for a hapten-specific receptor on T lymphocytes.

REJECTION OF ASCITES TUMOR ALLOGRAFTS

A pathway for cell-mediated tumor destruction in vitro by immune peritoneal exudate lymphoid cells has been proposed. The union of lymphocytes and tumor cells precedes the formation of an intermediate phase leading to lysis. The initial interaction is only partially temperature dependent. The cytolytic process per se is highly temperature dependent, being negligible at 25 degrees C but proceeding rapidly at 37 degrees C. (51)Cr release from tumor cells is demonstrable within 10 min at 37 degrees C and can be reversibly arrested by cooling. Once initiated, lysis is largely independent of additional interactions and continues at almost full rate for 30 min. The effector cells are not lysed and appear to be free to enter into further effector cycles.