Peritoneal Exudate Cell Population Research Articles

The Anti-apoptotic Murine Cytomegalovirus Protein vMIA-m38.5 Induces Mast Cell Degranulation.

Mast cells (MC) represent “inbetweeners” of the immune system in that they are part of innate immunity by acting as first-line sentinels for environmental antigens but also provide a link to adaptive immunity by secretion of chemokines that recruit CD8 T cells to organ sites of infection. An interrelationship between MC and cytomegalovirus (CMV) has been a blank area in science until recently when the murine model revealed a role for MC in the resolution of pulmonary infection by murine CMV (mCMV). As to the mechanism, MC were identified as a target cell type of mCMV. Infected MC degranulate and synthesize the CC-chemokine ligand-5 (CCL-5), which is released to attract protective virus-specific CD8 T cells to infected host tissue for confining and eventually resolving the productive, cytopathogenic infection. In a step forward in our understanding of how mCMV infection of MC triggers their degranulation, we document here a critical role for the mCMV m38.5 gene product, a mitochondria-localized inhibitor of apoptosis (vMIA). We show an involvement of mCMV vMIA-m38.5 in MC degranulation by two reciprocal approaches: first, by enhanced degranulation after m38.5 gene transfection of bone marrow-derived cell culture-grown MC (BMMC) and, second, by reduced degranulation of MC in peritoneal exudate cell populations infected ex corpore or in corpore with mutant virus mCMV-Δm38.5. These studies thus reveal a so far unknown function of mCMV vMIA-m38.5 and offer a previously unconsidered but biologically relevant cell system for further analyzing functional analogies between vMIAs of different CMV species.

Phagocytosis in Mesocestoides vogae-induced peritoneal monocytes/macrophages via opsonin-dependent or independent pathways

Summary Intraperitoneal infection with larvae of cestode Mesocestoides vogae offers the opportunity to study dynamic changes in the proportion and functions of individual cell types under a direct influence of parasites. The phagocytic activity is one of the basic effector functions of professional phagocytes and receptor-mediated uptake is a central in implementation of inflammatory responses. Present study extends information on this issue by exploring several phagocytosis pathways in M. vogae-induced myelo-monocytic cells. In addition, we analyzed proportions of morphologically distinct phenotypes within macrophage compartments after oral inoculation of larvae to mice. In gradually elevated population of peritoneal exudate cells, monocytes/ macrophages and giant cell were dominant cell types from day 21 p.i. Phagocytic activity of these cells had biphasic behaviour for both opsonin-dependent and independent pathways, whereas uptake by multinucleated macrophages was profoundly reduced. Highly elevated proportions of activated phagocytic cells were found from day 7 to 14 p.i., regardless particle type (latex beads, HEMA, liposomes) and opsonisation. Source of opsonins used for coating of liposomes suggested higher expression of complement receptors than Fc receptors on these cells, although the uptake of non-opsonized liposomes had different kinetics and was very high by activated cells early p.i. Present data indicate that early recruited macrophages/monocytes attain pro-inflammatory functions as indicated by highly elevated phagocytosis of immunologically inert particles as well as opsonized liposomes what is down-regulated once larvae start to proliferate in the peritoneal cavity, suggesting the role of parasite-derived molecules in modulation of this key phagocytes function.

Alternatively Activated Macrophages Elicited by Helminth Infection Can Be Reprogrammed to Enable Microbial Killing

The prime function of classically activated macrophages (activated by Th1-type signals, such as IFN-gamma) is microbial destruction. Alternatively activated macrophages (activated by Th2 cytokines, such as IL-4 and IL-13) play important roles in allergy and responses to helminth infection. We utilize a murine model of filarial infection, in which adult nematodes are surgically implanted into the peritoneal cavity of mice, as an in vivo source of alternatively activated macrophages. At 3 wk postinfection, the peritoneal exudate cell population is dominated by macrophages, termed nematode-elicited macrophages (NeMphi), that display IL-4-dependent features such as the expression of arginase 1, RELM-alpha (resistin-like molecule alpha), and Ym1. Since increasing evidence suggests that macrophages show functional adaptivity, the response of NeMphi to proinflammatory Th1-activating signals was investigated to determine whether a switch between alternative and classical activation could occur in macrophages differentiated in an in vivo infection setting. Despite the long-term exposure to Th2 cytokines and antiinflammatory signals in vivo, we found that NeMphi were not terminally differentiated but could develop a more classically activated phenotype in response to LPS and IFN-gamma. This was reflected by a switch in the enzymatic pathway for arginine metabolism from arginase to inducible NO synthase and the reduced expression of RELM-alpha and Ym1. Furthermore, this enabled NeMphi to become antimicrobial, as LPS/IFN-gamma-treated NeMphi produced NO that mediated killing of Leishmania mexicana. However, the adaptation to antimicrobial function did not extend to key regulatory pathways, such as IL-12 production, which remained unaltered.

Swimming training prevents generation of suppressor macrophages during acute cold stress

Acute cold stress induces suppressor macrophages expressing large numbers of receptors to Fc portion of immunoglobulin G (MAC-1+ Fc gammaRII/IIIbright cells), resulting in suppression of splenocyte mitogenesis. The generation of MAC-1+ Fc gammaRII/IIIbright cells is partly mediated by increased glucocorticoid levels during acute cold stress. The aim of the current study was to investigate the effect of swimming training on the generation of the MAC-1+ Fc gammaRII/IIIbright suppressor macrophages by acute cold stress. The trained mice underwent a 6-wk endurance swimming training (5 times/wk) in water at 35-36 degrees C for 90 min. The swimming training significantly increased brown adipose tissue mass, suggesting improved cold tolerance. Actually, when the swimming-trained mice were exposed to 5 degrees C for 3 h (acute cold stress), the rectal temperature was not decreased. The proportion of MAC-1+ Fc gammaRII/IIIbright cells in peritoneal exudate cells from swimming-trained mice was significantly lower than that from control mice. In addition, the proportion of MAC-1+ Fc gammaRII/IIIbright cells in peritoneal exudate cell population from swimming-trained mice was unaffected by the acute cold stress. The swimming training significantly attenuated the increases in serum corticosterone levels in response to acute cold stress. These results suggested that swimming training not only improves cold tolerance but also inhibits the generation of suppressor macrophages under acute cold stress as well as under normal conditions.

Requirement for In Vivo Production of IL-4, But Not IL-10, in the Induction of Proliferative Suppression by Filarial Parasites

Loss of T lymphocyte proliferation and the emergence of a host response that is dominated by a Th2-type profile are well-established features of human filariasis. We have previously reported that adherent peritoneal exudate cells (PEC) from mice transplanted with adult Brugia malayi parasites suppress the proliferation of lymphocytes without blocking Ag-cytokine production in vitro. We now show that infection of mice with the infective larval (L3) stage of B. malayi generates a similar population of PEC. Suppressive cells are generated within 7 days of infection and mediate their effects through a nitric oxide-independent pathway. Both L3 and adult infection elicit high levels of host IL-4 whereas the microfilarial stage of the parasite induces IFN-gamma production and does not generate a similar form of suppression. Production of host IL-4 was necessary to allow the generation of suppressive PEC, given that IL-4-deficient mice implanted with adult parasites failed to induce proliferative block. However, IL-10-deficient mice implanted with adult parasites resulted in T cell suppression, indicating that IL-10 is not essential for the induction of hyporesponsiveness. Neither IL-4 nor IL-10 were directly responsible for ablating cellular proliferation in vitro, as the addition of neutralizing Ab to either cytokine did not reverse the proliferative block. Thus, IL-4 produced in vivo in response to filarial L3 and adult parasites is essential for the induction of proliferative suppression but is not itself the suppressive factor.

Requirement for In Vivo Production of IL-4, But Not IL-10, in the Induction of Proliferative Suppression by Filarial Parasites

Loss of T lymphocyte proliferation and the emergence of a host response that is dominated by a Th2-type profile are well-established features of human filariasis. We have previously reported that adherent peritoneal exudate cells (PEC) from mice transplanted with adult Brugia malayi parasites suppress the proliferation of lymphocytes without blocking Ag-cytokine production in vitro. We now show that infection of mice with the infective larval (L3) stage of B. malayi generates a similar population of PEC. Suppressive cells are generated within 7 days of infection and mediate their effects through a nitric oxide-independent pathway. Both L3 and adult infection elicit high levels of host IL-4 whereas the microfilarial stage of the parasite induces IFN-gamma production and does not generate a similar form of suppression. Production of host IL-4 was necessary to allow the generation of suppressive PEC, given that IL-4-deficient mice implanted with adult parasites failed to induce proliferative block. However, IL-10-deficient mice implanted with adult parasites resulted in T cell suppression, indicating that IL-10 is not essential for the induction of hyporesponsiveness. Neither IL-4 nor IL-10 were directly responsible for ablating cellular proliferation in vitro, as the addition of neutralizing Ab to either cytokine did not reverse the proliferative block. Thus, IL-4 produced in vivo in response to filarial L3 and adult parasites is essential for the induction of proliferative suppression but is not itself the suppressive factor.

Relationship between cold tolerance and generation of suppressor macrophages during acute cold stress.

Acute cold stress induces suppressor macrophages expressing large numbers of receptors to the crystallizable fragment (Fc) portion of immunoglobulin G (MAC-1+ FcgammaRII/IIIbright cells), resulting in the immunosuppression of splenocyte mitogenesis. The generation of MAC-1+ FcgammaRII/IIIbright cells is mediated by the action of glucocorticoids (GCs) through the GC-receptor. In the present study, the generation of MAC-1+ FcgammaRII/IIIbright cells in peritoneal exudate cells was closely related to the decrease of rectal temperature during 3-day exposure to 5 degrees C. We next investigated the effects of improved cold tolerance on the generation of MAC-1+ FcgammaRII/IIIbright cells during acute cold stress. Mice were adapted to cold by exposure to 5 degrees C for 3 wk (cold-acclimated mice) and then reexposed to 5 degrees C for 3 h (acute cold stress) after living at 25 degrees C for 24 h. The rectal temperature of cold-acclimated mice was not decreased by the acute cold stress. In addition, the proportion of MAC-1+ FcgammaRII/IIIbright cells in peritoneal exudate cell population from cold-acclimated mice was unaffected by the acute cold stress. The cold acclimation significantly attenuated the increases in serum corticosterone levels and the expression of the GC-receptor mRNA on peritoneal exudate cells in response to acute cold stress. These results suggest that the altered GC response to acute cold stress by the improvement of cold tolerance inhibits the generation of suppressor macrophages during acute cold stress.

Responsiveness of susceptible inbred mice to Yersinia pseudotuberculosis Serovar III Infection

The susceptibility of BALB/c, C57BL and BDF1-hybrid mouse strains to Yersinia pseudotuberculosis serovar III infection was studied. The bacterial load in the viscera and brain and the host responses at different levels, i.e. blood, peritoneal cavity and organs were determined. Blood cell parameters and peritoneal exudate cell population were evaluated during the infection using the automated hematologic analyzer Technicon H-1. It was found that BDF1-hybrid mice produced an early peritoneal inflammatory response, while in BALB/c and C57BL mice it was not observed. The high susceptibility of C57BL was associated with a great number of microorganisms in the organs and with the corresponding histological changes. It was shown that the magnitude of the inflammation induced by Y. pseudotuberculosis varied among the host strains used. The variations of the susceptibility to Y. pseudotuberculosis among inbred mouse strains suggest the possible role of genetic factors regulating the host defence.

The mechanism of action of a synthetic immunomodulator, 3,6-Bis(2-piperidinoethoxy)acridine trihydrochloride (CL 246, 738), in natural killer cell activation in animals

CL 246,738 is a low molecular weight, synthetic immunomodulator. The present study was done to determine the interaction among interferon (IFN), macrophages, and natural killer (NK) cells in mice following oral administration of CL 246,738. Splenic NK activity as evidenced by lysis of YAC-1 lymphoma cells in vitro was found to be augmented by the compound not only in normal mice, but also in immunodeficient beige and nude mice. Lytic activity remained elevated from one to seven days after a single treatment and the peak activation varied depending on the source of NK cells. NK cell activity associated with the peritoneal exudate cell population peaked at day 1 and returned to normal by day 2, whereas NK cell activity of peripheral blood lymphocytes peaked at day 3 and remained significantly elevated until day 7. Liver associated NK activity peaked at day 4 and remained significantly elevated at day 7 after treatment with CL 246, 738. Lung associated NK activity was elevated by day 1 after treatment, peaked at day 4 and returned to normal by day 7 after drug administration. The drug was also effective in inducing IFN in all mouse strains tested. When these drug-treated mice were given antibody to IFN-(α + β) but not to IFN-(β), both IFN levels and NK cell activity decreased, suggesting the importance of IFN-(α) in this system. Furthermore, mice that had received carrageenan prior to, but not after CL 246,738 administration showed reduced serum IFN titers as well as decreased NK cell activity, indicating that macrophages played an intermediate role in immune enhancement by the drug. Taken together, these studies indicate that the mechanism of action of CL 246,738 in vivo is to induce macrophages to release IFN-(α) which subsequently activates NK cells.

Perforin mRNA in primary peritoneal exudate cytotoxic T lymphocytes.

Considerable evidence indicates that cloned CTL cell lines kill target cells by releasing toxic granules that contain a cytolytic protein, called perforin, and several serine esterases (granzymes A to F). However, primary CTL, such as the highly cytolytic peritoneal exudate lymphocyte (PEL) cell population, have been found by a hemolytic assay to have no perforin, or perhaps only borderline levels of that protein, suggesting that these cells use a different lytic mechanism. To determine whether or not primary CTL express the perforin gene, we have here compared mRNA from PEL CTL and from a cloned CTL cell line, 2C, by Northern blot analysis using a perforin cDNA probe. CD8+ PEL CTL contain approximately 30% of the amount of perforin message present in 2C. Moreover, depletion of CD8+ T cells from the total peritoneal exudate cell population removes both cytolytic activity and perforin message. We have previously shown that PEL CTL elicit the same changes in target cells as cloned CTL cell lines and are resistant to lysis by the toxic granules purified from these cells lines. Taken together these results are consistent with the view that primary CTL, as well as long term cloned CTL cell lines, exercise their cytolytic activity by means of perforin.

Chemotactic effect of human recombinant interleukin 2 on mouse activated large granular lymphocytes.

Human recombinant interleukin 2 (hrIL-2) was demonstrated in vitro to be chemotactic for mouse large granular lymphocytes (LGL) activated in vivo by virus infection. Peritoneal exudate cells harvested from virus-infected mice were used as a source of LGL. LGL collected from mouse hepatitis virus-infected mice at 3 days postinfection were a source for NK 1.1 positive natural killer (NK)/LGL. LGL collected from mice treated with antiserum to gangliotetraosylceramide and infected with lymphocytic choriomeningitis virus for 7 days were used as a source for Lyt-2 positive cytotoxic T lymphocytes (CTL)/LGL. Both NK/LGL and CTL/LGL responded chemotactically to hrIL-2, purified IFN-beta, and to crude cell-free washout fluids collected from the peritoneal cavity of virus-infected mice. hrIL-2 had chemotactic activity for virus-elicited granular and agranular lymphocytes but did not attract the contaminating macrophages, in contrast to IFN-beta, which displayed chemotactic activity for virus-elicited granular and agranular lymphocytes as well as macrophages. The migration to hrIL-2 was inhibited by a monoclonal antibody (7D4) to the IL-2 receptor, but treatment with 7D4 did not affect migration in response to IFN-beta. Microscopic examination of Wright's-Giemsa-stained migrated NK/LGL and CTL/LGL revealed that the majority of migrated LGL in either LGL population had a blast cell morphology (enlarged cells with rich basophilic cytoplasm). The frequency of cells bearing the LGL morphology within the virus-elicited nonadherent peritoneal exudate cell population was on incubation in vitro, stabilized by either hrIL-2 or IFN-beta. These data suggest that another important immunomodulating function of IL-2 may be to attract activated NK/LGL and CTL/LGL to sites of inflammation.

Vaccinia virus proteins on the plasma membrane of infected cells III. Infection of peritoneal macrophages

Primary macrophage cultures were prepared from the peritoneal exudate cell population harvested from mice challenged intraperitoneally with saline, thioglycollate, or vaccinia virus. Vaccinia virus was adsorbed and penetrated into primary macrophages and L-cells with similar kinetics. As evidenced by the expression of some “early” virus-specified proteins, partial uncoating and activation of the virion-associated DNA-dependent RNA polymerase occurred in the infected macrophages. Subsequently, the viral replication cycle in macrophages was aborted; with time after infection, viral DNA and virion proteins initially associated with infected cells could be detected in an acid-soluble form in the medium harvested from infected macrophage cultures. The results suggest that at the time that the final stages of virus uncoating should have occurred, intracellular subviral particles were, instead, degraded in the infected, primary macrophages. Viral DNA synthesis could not be measured in vaccinia virus-infected macrophages, no “late” virus functions were expressed, and progeny virions were not assembled. As measured by the binding of antiviral-antibody- 125I-protein A complexes to the surface of vaccinia virus-infected cells, the expression of virus-specified antigens on the surfaces of infected macrophages was significantly reduced and never exceeded that measured at 2 hr after infection on the surfaces of infected L-cells. The expression of virus-specified polypeptides with mol mass of 48–50, 45–46, 36–37, and 25 kDa on the plasma membranes of vaccinia virus-infected, thioglycollate-elicited macrophages, rendered the infected macrophages susceptible to lysis by vaccinia virus-specific cytotoxic T-cells.

Tumor--host interactions in vivo. II. Induction of NK-cell activity by intraperitoneal injection of murine sarcoma cells or tumor ascites fluid.

We have earlier shown that coculture of macrophages and cells from a methylcholanthiene‐induced sarcoma (MCI M‐AA) in vitro resulted in macrophage activation and production of type II (gamma) interferon. When ascites fluid from the MCIM‐AA sarcoma (shown previously to activate macrophages in vitro) was added to spleen cell populations from semisyngeneic C3D2 mice in vitro. NK activity was markedly enhanced. After intraperitoneal injection of MCI M‐AA cells or ascites fluid, 4‐ to 12‐fold increased NK cell activity with a peak at 3–5 days could be measured in the spleen cell population and in the non‐adherent peritoneal exudate cell population. The mice injected with tumour cells or ascites fluid developed a pronounced splenomegaly, and maximum spleen size coincided with peak NK activity. Injection of tumour cells or tumour ascites fluid resulted in marked changes in the T‐cell, B‐cell, macrophage, and ‘null’ cell content of ihe peritoneal exudate.

Macrophages, Langerhans cells, interdigitating and dendritic accessory cells: a summary.

Adherent or accessory cells, previously indicated as “A”-cells are of paramount importance in the immune response in vitro as well as in vivo. The nonlymphoid adherent population of peritoneal exudate cells, which have phagocytic properties, comprise the accessory cells which have to be present in lymphoproliferative responses in vitro. Most of these cells were obviously macrophages. However, they constitute a heterogeneous population of cells. It appeared that only a minority (8–15%) of these cells bear the Ia determinants of the immune response gene complex (1) which determines their antigen presenting function. Moreover, macrophages may change their properties in this respect during adhesion in vitro (2).KeywordsAccessory CellMononuclear Phagocyte SystemAntigen Present FunctionPeripheral Lymphoid OrganBirbeck GranuleThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

In-Vivo Modulation of Macrophage Functions by Herpes Simplex Virus Type 2 in Resistant and Sensitive Inbred Mouse Strains

Intra-peritoneal (i.p.) infection of mice with herpes simplex virus type 2 (HSV 2) attracted macrophages into the peritoneum. Macrophages from moderately and highly HSV 2 resistant mouse strains expressed elevated phagocytosis activity 24 hours after injection. Stimulation of phagocytosis in low resistant strains was generally less effective or absent. This was, in some experiments, due to the fact that macrophages were already highly activated before the experimental infection. I.p. infection also caused HSV replication in the adherent peritoneal exudate cell (PEC) population. The capacity of macrophages supporting HSV 2 replication was low in three of four resistant mouse strains and high in all moderately and highly susceptible and in one of the resistant (SJL) strains when determined 24 hours after infection. Four different F1 hybrids between resistant and susceptible strains exhibited significantly lower yields of virus-producing macrophages than the HSV-sensitive parent. One hybrid between two HSV-susceptible lines restricted virus replication in the PEC population better than both parental strains.

The Cellular Stimuli for the Rejection of Established Islet Allografts

Previous work from our laboratory has indicated that the transplantation of pancreatic islets is a feasible approach to the problem of diabetes. A major obstacle to transplantation is presented by passenger leucocytes, which contaminate the preparations and can lead to the prompt rejection of fresh islets. We have extended our previous studies on the rejection of islet allografts by challenging transplanted animals with enriched lymphoid cell populations prepared from animals both syngeneic to the transplanted islets and third party. Rapid and complete rejection was observed when the challenge peritoneal exudate cell population was syngeneic with the transplanted islets; rejection was determined by both functional and histologic criteria. Peritoneal exudate cells from a third-party rat strain induced delayed and variable effects upon the function of the transplant. In contrast, splenic T-cells were capable of inducing rejection, regardless of the strain of origin, though the time course of T-cell-induced rejection was slower than that observed by syngeneic peritoneal exudate cells. Finally, splenic B-cells completely failed to induce rejection. Our data indicate that at least two mechanisms exist by which the rejection of islet allografts may be triggered. The first is a haplotype-specific mechanism initiated by a cell type present at high frequency in peritoneal exudate cells; these are probably macrophages. The second mechanism is initiated by immunocompetent T-cells; this mechanism shows no haplotype specificity. We suggest that both macrophages and T-cells must be considered when devising protocols for the removal of passenger leucocytes from allografts.

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.

Impairment of antigen-presenting cell function by ultraviolet radiation.

UV light irradiation of BALB/c mice was found to result in impairment of antigen-presenting cell function. Adherent trinitrophenyl-derivatized cells from the peritoneal exudate cell population or the spleen of UV-treated donors could not induce hapten-specific delayed hypersensitivity responses in UV-irradiated syngeneic mice, whereas adherent trinitrophenyl-derivatized cells from normal mice were able to do so. The failure to induce immunity in UV-treated mice by utilizing UV-treated adherent antigen-presenting cells was associated with the development of antigen-specific suppressor T cells. The implication of these results for UV-induced carcinogenesis is discussed.