Comparison of locomotion, chemotaxis and adhesiveness of rabbit neutrophils from blood and peritoneal exudates

We compared the spontaneous behaviour (motility, adhesiveness, locomotion) and the chemotactic responses of exudate and blood-borne neutrophils. Directional locomotion of exudate neutrophils in 2% HSA-Gey's towards exudate fluid was not significantly changed, the response to activated autologous plasma diminished, and that to f-Met-Leu-Phe (10(-9) M) increased in comparison with blood-borne cells. The spontaneous behaviour of exudate cells in 2% HSA-Gey's (no gradient) differed markedly from that of blood-borne cells. In tissue culture medium (2% HSA-Gey's) exudate cells showed heightened motility in suspension and greater adhesiveness to glass substrata. These differences were eliminated by culturing the cells in their physiological media (i.e. plasma or exudate fluid). In contrast to blood-borne cells, exudate neutrophils tended to aggregate spontaneously. There was no correlation between neutrophil aggregation and adhesion to glass substrata of exudate cells in exudate fluid.     

In situ activation of syngeneic tumour-specific cytotoxic T lymphocytes: Intra-pinna immunization followed by restimulation in the peritoneal cavity

Summary Tumour-specific cytotoxic T lymphocytes (CTL) are usually obtained after immunization in vivo and restimulation of immune cells in vitro. We here describe the generation of syngeneic tumour-specific CTL within no more than 9 days by priming and restimulation in vivo. This is achieved only if the correct sites are used both for primary immunization (ear pinna) and for restimulation (peritoneal cavity). The kinetics of immune T cell induction and of the secondary response in vivo will be reported. While a secondary CTL response could be generated in the peritoneal cavity, this was not possible in the spleen, no matter which routes of antigen restimulation were used. Upon transfer of immune spleen cells into the peritoneal cavity but not into the spleen, a secondary response could be generated upon in situ restimulation, indicating the importance of the correct microenvironment for this type of response. The peritoneal effector cells were true T cells and recognized a tumour-associated antigen in association with the K^d major histocompatibility (MHC class I) antigen. Finally the activated tumour-specific peritoneal exudate cells were able to transfer protective immunity without exogenous interleukin-2 into normal syngeneic mice.     

Transfer Agent of Immunity

Detection of the cells which contain antibody was accomplished by a method of immune adherence of human erythrocytes to a single cell, termed SCIA (single cell immune adherence) reaction. Peritoneal exudate cells were collected from mice immunized with flagella of either Salmonella enteritidis or S. tennessee. Serologically specific antibody was detectable in some of the peritoneal exudate cells of such mice. An immune ribonucleic acid (RNA) was extracted from the peritoneal exudate cells of mice immunized with salmonella flagella. When mice were injected intraperitoneally with this preparation, serologically specific antibody was found in some of their peritoneal exudate cells by the SCIA method. This preparation was inactivated by treatment with ribonuclease, but was resistant to proteinases, deoxyribonuclease and anti-flagella antibody, suggesting that this agent is of RNA nature and does not contain antigen or fragment thereof.     

The immune system response to antigen in cold- and warm-adapted rats

In rat experiments, it was shown that long-term adaptation to cold modified the response pattern of the immune system to antigen stimulus by variously changing the different components of the response. There was a considerable decrease in the number of antigen-binding cells in spleen and peritoneal exudate and also a marked decrease in general antibodies in blood. Conversely, the number of antibody-forming cells in spleen increased twofold. The shift in the thermal conditions after immunization caused changes in the immune response. After the placement of cold-adapted animals to the warm environments the number of antigen binds and antibody-forming cells in spleen, as well as hemagglutinin titers in serum normalized, became indistinguishable from those in the warm-adapted control group. The placement from warm to cold after immunization of warm-adapted rats also produced considerable shifts in parameters of the immune response. However, these shifts were not entirely consistent with those observed for long-term adapted to cold. The obtained data evidence that the thermal factor of the environment is of major importance to the function of immunocompetent cells.     

Electron microscopic studies on the peritoneal exudate of the mouse to determine the effect of colloidal carbon on the primary immune response to heterologous erythrocytes]

Antibody production to sheep erythrocytes was markedly depressed in mice injected intraperitoneally with colloidal carbon. Peritoneal exudate cells were investigated electron microscopically with regard to mechanisms responsible for lower antibody production. Peritoneal macrophages were not disturbed in antigen phagocytosis and antigen digestion. Only in connection with immunosuppression was there an increase in antigen containing macrophages in the peritoneal cavity. The changing relation of antigen-stimulated macrophages and lymphocytes in favour of the macrophages and its consequence to decreased immune response is discussed.     

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.     

Correlation of immunogenicity and production of ornithine by peritoneal macrophages

The release of ornithine by macrophages and its correlation with their immunogenicity after treatment with various macrophage-stimulating substances were analyzed. Pristane-elicited peritoneal macrophages (PM) were found to express strong arginase activity and to release L-ornithine into the extracellular space. This activity is strongly reduced within 3 hr after treatment with tetradecanoylphorbol acetate (TPA) but not with lipopolysaccharide (LPS). Resident PM usually express little arginase activity, but this activity is markedly augmented within 24 or 48 hr after treatment with LPS. The release of ornithine by peritoneal cells (PC) (60 to 90% macrophages) was found to be correlated with their immunogenicity as determined by the in vivo immunization for a subsequent in vitro secondary cytotoxic response against minor H antigens. The immunogenicity of pristane-elicited PC is markedly stronger than that of resident PC or TPA-treated, pristane-elicited PC. Moreover, the immunogenicity of the resident PC and TPA-treated elicited PC is substantially augmented by the simultaneous injection of ornithine, whereas the immunogenicity of the untreated elicited PC is not further augmented by exogenous ornithine, indicating that the endogenous production of ornithine by the stimulating cells had a strong influence on the resulting immune response. Injection of glutathione into pristane-treated mice also reduces the ornithine production and immunogenicity of the resulting peritoneal exudate cells. The immunogenicity in this case is at least partly reconstituted by application of exogenous ornithine. Our experiments revealed no correlation between the production of ornithine and prostaglandin E2. Prostaglandin E2 production of resident and pristane-elicited PC is not markedly different and is in either case strongly augmented by TPA. Elicited or resident PM which have been incubated for several days in culture release practically no ornithine; but ornithine production can be induced again by incubation for 24 hr with LPS and to some extent also with interferon-gamma.     

Suppression of responses to cryptococcal antigen in murine cryptococcosis

Subpopulations of spleen cells responsible for responsiveness and unresponsiveness to cryptococcal antigen in vitro were identified. Lymphocytes which responded in lymphocyte transformation (LT) assays were nylon wool nonadherent and theta antigen positive. These lymphocytes required the presence of an accessory cell which could be supplied by normal peritoneal exudate cells. Spleen cells taken from mice which had been infected for 3 to 15 days were tested to determine their ability to respond to cryptococcal antigen in LT assays. A minimal response was detected at the ninth day of infection. The response of infected spleen cells was attributed to a nonadherent lymphocyte. Nonadherent spleen cells of infected animals had enhanced responses after removal of adherent cells and addition of normal peritoneal exudate cells. Suppressor cells were detected in the spleens of infected mice by the 12th day of infection and thereafter. A nonadherent suppressor cell was identified, but indirect evidence suggested that an adherent cell could also be present in infected spleens.     

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 X 10(7) normal PE cells, significant specific swelling was obtained using 1-3 X 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.     

Antigen presentation by liver sinusoidal lining cells after antigen exposure in vivo

The ability of liver sinusoidal lining cells (LSLC), a mixture of Kupffer cells and endothelial cells, to function as antigen-presenting cells (APC) was examined. Guinea pig LSLC were found to present antigen in vitro, albeit somewhat less effectively than a reference population of peritoneal exudate macrophages. The difference in APC function could not be explained by a deficiency of interleukin 1 (IL 1), as LSLC secreted IL 1 and expressed membrane-bound thymocyte stimulatory activity. The ability of LSLC to take up antigen from the portal blood in vivo and present it to primed T lymphocytes in vitro was also investigated. Trinitrophenyl-ovalbumin was injected intraportally into either strain 13 or strain 2 guinea pigs. The LSLC were subsequently isolated by collagenase digestion and density separation and assessed for the ability to induce proliferation of antigen-primed accessory cell-depleted syngeneic peritoneal exudate T lymphocytes in vitro. The in vivo antigen-pulsed LSLC were found to present antigen in vitro to primed T cells in an antigen-specific and genetically restricted manner. T cell DNA synthesis induced by antigen-bearing LSLC could be augmented by coculture with additional accessory cells, but not IL 1-containing macrophage supernatants. Enhancement of responsiveness was not genetically restricted. The demonstration that LSLC can take up, process, and retain antigen in vivo and present it to primed T cells in vitro suggests that LSLC are capable of contributing to the immune response to antigens appearing in portal blood.     

The nature of the factors in immune lymph nodes that stimulate humoral and cellular immune reactions

In the early period after antigen action, cells of the lymph node draining area of antigen injection produce a factor, which after its administration to intact mice, promotes intensified migration of myelopeptide producers in the bone marrow and increases bone marrow suppressor activity. Factor administration produced 2-3-fold increase in macrophage, peritoneal and lymph node count as compared with control, and 2-4-fold increase in humoral and cellular immune response. The factor consists of three easily identifiable components: Ig, MHC class II determinants and antigen determinants, that form unique complex similar to, or identical with the complexes detected in vivo within 3-6 hours after immunization in serum by other authors.     

Changes in Glutathione Peroxidase Activities and the Oxidative Burst of Leukocytes During Inflammation in the Mouse and Rat

The relationship between glutathione peroxidase (GSH-Px) activity and opsonized zymosan-induced chemiluminescence (CL) has been studied with exudate leukocytes obtained at different times after induction of inflammatory responses in the mouse peritoneal cavity with heat-killed Corynebacterium parvum and in the rat pleural cavity with I-carrageenin. GSH-Px activity in mouse peritoneal exudate cells fell markedly after 2–4h, returning to normal within 1–2 days. The lowered enzyme activity was associated with an increased ability of the cells to generate CL. Rat pleural exudate cells exhibited a slight fall in GSH-Px activity after 6h which increased to supranormal levels within 1–2 days. During this period the ability of the cells to generate CL continually increased. The data indicate that during the early phase of increased generation of reactive oxygen species (ROS) by inflammatory leukocytes, the intracellular protective mechanism, represented by GSH-Px, is compromised. Subsequently, GSH-Px activity increases to or above initial levels possibly due to the presence of mononuclear cells and/or as a response to the increased generation of ROS.     

Prostaglandin-mediated suppression of delayed-type hypersensitivity to infected erythrocytes during Babesia microti infection in mice

The mechanism of suppression of delayed-type hypersensitivity (DTH) to intraerythrocytic Babesia microti which occurs during infection in mice was examined. The suppression was not specific for anti-parasite DTH; infected mice immunized and challenged with sheep red blood cells had a similar depression of anti-sheep red blood cell DTH. Sublethal or lethal irradiation did not significantly alter the suppression of the DTH response, and cyclophosphamide pretreatment of infected mice also had no effect on suppression. Multiple passive transfer experiments using serum or regional lymph node cells from immunized or infected and immunized (suppressed) donor animals failed to demonstrate any ability to transfer suppression of DTH. Adherent cells from the spleens or peritoneal exudates of suppressed mice, however, did significantly depress the ability of immunized mice to express a DTH response. The cells responsible for this suppression were Thy 1- and nonspecific esterase+. Treatment of suppressive cell populations with 10 micrograms/ml indomethacin for 24 hr in vitro abrogated their suppressive ability, and in vivo administration of indomethacin to suppressed mice also restored DTH to normal levels. By examining levels of prostaglandin E2 (PGE2) in supernates of cultured peritoneal exudate cells from immune or suppressed mice, it was shown that infected mice had peritoneal exudate cells which produced significantly more PGE2 than similar cells from immune mice. These data suggest that B. microti infection elicits synthesis of PGE2 by macrophage-like cells which results in suppression of DTH to parasite as well as heterologous antigens.     

The chemiluminescence reactions of the blood neutrophils and of peritoneal exudate cells in Syrian hamsters to the intraperitoneal administration of cellular and microbial materials]

The luminol-dependent chemiluminescence (CL) activity of peritoneal exudate cells and blood neutrophils of Syrian hamsters inoculated intraperitoneally with heat-inactivated microbial particles of Candida albicans, (C. albicans), heated irradiated normal cells and native or heated irradiated malignant tumor cells was studied. The inoculation with particles of C. albicans and heated normal cells induced significant activation of CL of peritoneal exudate cells, but did not influence the CL reaction of blood neutrophils. The inoculation of animals with nonheated irradiated tumor cells led to increase of CL response of both peritoneal exudate cells and blood neutrophils. The inoculation with heated irradiated tumor cells did not activate CL of peritoneal exudate cells and led to slight, but long-lasting decrease of CL response of blood neutrophils.     

Immunomodulating action of peptidoglycan of microbial origin]

The action of a high molecular weight peptidoglycan produced by Agrobacter radiobacter sp. on the functional activity parameters in leukocytes and macrophages i. e. chemotaxis and adhesion was studied. It was shown that the peptidoglycan had a stimulating action on the chemotaxis of cells of the peritoneal exudate. A marked stimulating action of the drug on the primary immune response to the tissue antigen of sheep erythrocytes was observed. The peptidoglycan stimulated the antibody titers and delayed hypersensitivity when administered in various periods after an antigenic stimulus. Multifactorial experiments on the protective action of the peptidoglycan in experimental infections were carried out. Second-order polynomial statistic models characterizing the animal survival rate were constructed and the dose-time parameters of the drug use were optimized.     

Target cells for the activity of a synthetic adjuvant: muramyl dipeptide.

Muramyl dipeptide (MDP), a synthetic adjuvant, increased the primary response of CBA mice to sheep red blood cells (SRBC). In reconstituted irradiated recipients, cooperation between T and B lymphocytes was required for the expression of adjuvant activity and MDP increased the efficiency of SRBC-educated T cells. The role of T-derived lymphocytes in mediating the MDP adjuvant activity was also demonstrated in irradiated mice and in mice reconstituted with various splenic cellular types of donors which had received SRBC and MDP 24 hr earlier. In our experiments, the macrophage did not seem to be involved, since MDP did not increase the phagocytic capacity of peritoneal exudate cells and MDP- and SRBC-pretreated macrophages had no increased ability to induce an anti-SRBC immune response. These results demonstrate the importance of T lymphocytes as mediators of the adjuvant activity of MDP.     

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.     

Induction of nonspecific killer cells by delayed-type hypersensitivity against soluble protein antigens in murine peritoneal cavities

Summary We induced nonspecific killer cells in the local site of delayed-type hypersensitivity against keyhole limpet hemocyanin or ovalbumin. Delayed-type hypersensitivity was induced in the peritoneal cavities of mice, and peritoneal exudate cells (PEC) were collected. These PEC were found to have killer activity toward SP2 and YAC-1 cells (target cells susceptible to natural killer cells) by 4-h ⁵¹Cr-release assays. The induction of killer activity in PEC was observed in parallel with the eliciting of delayed-type hypersensitivity in the peritoneal cavity, in which the killer activity was maximum 24–48 h after the antigen challenge, but was not induced in nu/nu mice and was induced in an antigen-specific way. These killer cells did not adhere to nylon wool and had Thy1 and asialo-GM1 antigens on their surfaces. Their precursor cells were also asialo-GM1-positive. These findings indicate that the killer cells probably belong to the NK cell lineage. Results of tumor challenge experiments showed that these killer cells had an antitumor effect in vivo as well as in vitro.     

The activation of guinea pig T lymphocytes by anti-beta 2-microglobulin serum.

In order to study further the role of beta 2-m in the regulation of the immune response, we have examined the effects of a goat anti-guinea pig beta 2-m serum on a number of T lymphocyte functions in vitro. Anti-beta 2-m serum produced a marked inhibition of the response of peritoneal exudate T cells to antigen and mitogen stimulation. Surprisingly, a marked activation of lymph node T lymphocyte proliferation was observed in the absence of antigen or mitogen stimulation. This stimulatory effect of anti-beta 2-m serum was shown to be specific for beta 2-m and required the presence of macrophages. The T cell proliferative response induced by anti-beta 2-m could not be blocked by antisera to the antigens of the guinea pig MHC. These studies suggest that beta2-m may play some critical role in the immune response at the level of T cell activation.     

Studies on T cell clonal expansion. II. The in vitro differentiation of pre-killer and memory T cells.

Spleen cells from C57BL/6 mice immunized with a DBA/2 mastocytoma (P815) were harvested at various stages of the immune response and cultured in vitro in the presence and absence of antigen. Killer T cell activity in immune spleens could not be demonstrated until 6 or 7 days after antigen, but spleen cells harvested as early as 3 or 4 days and cultured for 24 hr at 37 degrees C showed significant cytotoxicity. This increased activity was not augmented further by culturing with antigen. "Memory" T cells, whose in vitro differentiation into killer cells required the presence of antigen, could not be demonstrated until 9 or 10 days after alloantigenic stimulation. Once produced, however, these cells persisted for at least 6 months. Memory cells, like killer T cells bound avidly to homologous allogeneic monolayers. There were indications that the memory T cell pool was heterogeneous. On one hand, when cells harvested 10 days after stimulation were exposed to antigen in vitro, their lytic activity increased within 24 hr but showed no further increases when the culture period was extended. In contrast, 45-day-old immune cells showed increasing lytic activity throughout a 4-day exposure to antigen. Augmentation of lytic activity in both cell populations was independent of DNA synthesis through the first 24 hr of culture. Subsequent increases in the activity of 45-day cells was dependent upon cell proliferation. Both the antigen-independent augmentation of lytic activity which followed culturing of immune cells, and the antigen-induced differentiation of memory cells were reversibly inhibited by a series of drugs which raised lymphocyte cAMP levels.