Decreased phagocytosis by peritoneal macrophages from BCG-treated mice: induction of the phagocytic defect in normal macrophages with BCG in vitro.

Mice treated up to 31 weeks previously with intraperitoneal BCG yielded peritoneal macrophages with decreased phagocytosis of starch granules, latex beads, graphite dust and formalinized Listeria monocytogenes, with or without opsonin, compared to macrophages from untreated mice. These assays were selected to allow quantitative determinations of the rate or extent of particle uptake under nonrate-limiting conditions. Phagocytosis could be depressed to a similar degree by the prior addition of either starch granules or BCG to normal adherent peritoneal cell cultures in vitro. However, with these two particles, two different mechanisms of inhibition of subsequent phagocytosis appeared to be at work. Inhibition of phagocytosis by prior exposure to large amounts of starch granules appeared to consist largely of mechanical interference, as if through the preemption of intracellular space. In contrast, inhibition by prior uptake of BCG occurred with very small amounts of BCG and appeared gradually with time after uptake of BCG. The ability of a “macrophage activating agent” to inhibit selected functions of parasitized cells may help to explain some of the discordant results obtained by others studying phagocytosis by “activated” macrophages. Such agents may simultaneously enhance some macrophage functions and depress others.     

Suppression of in vitro maintenance and interferon-mediated augmentation of natural killer cell activity by adherent peritoneal cells from normal mice

The ability of adherent peritoneal cells (APC) to inhibit murine natural killer (NK) cell activity was examined. Nylon wool-nonadherent splenic effector cells were incubated overnight with or without different numbers of APC. NK activity was then measured against YAC-1 in a 4-hr 51Cr-release cytotoxicity assay. Proteose peptone-elicited or unstimulated resident APC from normal mice markedly suppressed NK activity of splenic effector cells in the presence or absence of exogenously added interferon. The suppression was dependent on the number of APC added with 10% APC, relative to the number of effector cells, resulting in a greater than 65% inhibition of cytotoxicity. The effector phase of cytotoxicity was not the target of the suppressor cells, because APC did not suppress NK activity when they were present only during the cytotoxicity assay. The addition of APC to alloimmune cytotoxic T cells under similar conditions resulted in no inhibition of cytotoxicity. Both syngeneic and allogeneic APC suppressed NK activity, but several murine macrophage-like cell lines lacked this property. In contrast to APC, incubation of effector cells with adherent spleen cells from normal mice resulted in no inhibition of NK activity. APC from mice injected with C. parvum were less inhibitory for NK activity than normal resident APC. In contrast, C. parvum APC suppressed concanavalin A-induced lymphoproliferation and were directly cytotoxic to tumor target cells in vitro, whereas normal APC lacked these properties. The results indicate that the peritoneum of untreated mice contains suppressor cells that can inhibit the in vitro maintenance and IFN-mediated augmentation of NK activity. In addition, these results indicate a broader spectrum of immune reactivities regulated by APC and suggest that, depending on their level of activation, APC can preferentially inhibit different immune functions.     

The destruction of allogeneic tumour cells by antibody and adherent cells from peritoneal cavities of mice.

Antisera to a DBA2 lymphoma (SL2) were raised in C57 black mice. The sera contained cell-dependent antibodies which lysed SL2 cells in conjunction with a monolayer of adherent peritoneal cells from unimmunised mice. The strongest lytic reaction was observed when the three components of the system, monolayer, target, and antiserum, were incubated together. The free antibody was not cytophilic for macrophages. It combined specifically with the target cell but precoated SL2 cells were not lysed effectively, probably because cells in the monolayer also accelerated the inactivation of antibody on the surface of the target cell.     

Clumping of Staphylococcus aureus in the peritoneal cavity of mice

Kapral, Frank A. (Philadelphia General Hospital, Philadelphia, Pa.). Clumping of Staphylococcus aureus in the peritoneal cavity of mice. J. Bacteriol. 92:1188-1195. 1966.-Nonencapsulated strains of Staphylococcus aureus inoculated into the peritoneal cavity of mice are promptly clumped by the interaction of fibrinogen with the bound coagulase present on the bacterial surface. Some of the pre-existing leukocytes adhere to the staphylococcal clumps during the 1st hr, but phagocytosis is minimal. During the 2nd hr, there is an influx of neutrophils into the region, and these form a thick layer around the staphylococcal clumps and, apparently, prevent further egress of toxin. Leukocytes in proximity to the organisms undergo degeneration, but cells located externally maintain an effective barrier and, thus, confine the organisms. The encapsulated Smith strain of S. aureus is not clumped under these circumstances, presumably because the capsule prevents the bound coagulase-fibrinogen interaction.     

Cytotoxic T cells in the peritoneal cavity of mice infected with ectromelia virus.

Peritoneal exudate cells from mice infected with ectromelia virus were cytotoxic for virus-infected target cells as measured in a 51Cr release assay. Cytotoxic activity seemed to be T cell-dependent as it was largely abolished by treatment with anti-theta serum and complement but was not impaired by macrophage depletion. The kinetics of development of cytotoxicity in the peritoneal cavity lagged behind spleen cytotoxicity by 1-2 days. Peak activity in peritoneal cells was present about 6 days after intravenous infection with virus. These studies suggest that macrophages present in the free peritoneal cell populations of ectromelia-infected mice are not cytotoxic for virus-infected target cells. The effect of macrophages in virus clearance is therefore likely to be due to phagocytic rather than cytotoxic effects.     

Characterization of glycosylation and adherent properties of melanoma cell lines

The repertoire of oligosaccharide components of cellular glycoproteins significantly contributes to cell adhesion and communication. In tumor cells, alteration in cellular glycosylation may play a key role in giving rise to invasive and metastatic potential. Over 100 melanoma cell lines deposited in the ESTDAB Melanoma Cell Bank (Tubingen, Germany) were studied for the characteristic glycan composition related to tumor progression. Analysis of: (1) cell adhesion to extracellular matrix proteins—fibronectin, laminin, and collagen; (2) the expression of selected glycosyltransferases—α2,3(Galβ1,3)- and α2,3(Galβ1,4)-sialyltransferases, α1,2- and α1,3-fucosyltransferases, and N-acetylglucosaminyltransferase V; (3) characterization of N-glycans was carried out on uveal (4), primary cutaneous (6), and metastatic (96) melanoma cell lines. Results showed that uveal cells did not adhere to any of the substrates and, in general, possessed less glycans containing α-2,6- and α-2,3-linked sialic acid. The average number of polypeptides bearing β-1,6-branched tri- and tetra antennary glycans(characteristic of the metastatic phenotype)were similar in uveal, primary cutaneous, and metastatic melanoma cell lines. Characterization of N-glycans may open a new perspective in the search for specific glycoproteins that could become targets for the therapeutic modulation of melanoma. This article is a symposium paper from the conference “Progress in Vaccination against Cancer 2004 (PIVAC 4)”, held in Freudenstadt-Lauterbad, Black Forest, Germany, on 22–25 September 2004     

Spontaneous cytotoxic activity of eosinophilic granule cells separated from the normal peritoneal cavity of Dicentrarchus labrax

In this study the spontaneous in vitro cytotoxic activity to tumour cell lines, (K562), by unstimulated sea bass (Dicentrarchus labrax) leukocytes was examined by trypan blue exclusion test and lactate dehydrogenase release assay. A high anti-tumour cell line activity of resident peritoneal leukocytes was found at an effector to target ratio (E:T) of 25:1 after incubation for 2 h at 18 degrees C. Rabbit and sheep erythrocytes were not lysed. A low activity was displayed by head kidney and spleen cell populations whereas blood leukocytes revealed no significant activity. The effect of E:T ratio on cytotoxicity as well as microscopy observations suggested that the cytotoxic reaction required effector-target cell contact. Eosinophilic granule cells, isolated on a Percoll density gradient from a peritoneal wash, appeared to be responsible for the in vitro cytotoxic activity.     

Neutrophils and monocytes transport tumor cell antigens from the peritoneal cavity to secondary lymphoid tissues

Antigen-transporting cells take up pathogens, and then migrate from sites of inflammation to secondary lymphoid tissues to induce an immune response. Among antigen-transporting cells, dendritic cells (DCs) are believed to be the most potent and professional antigen-presenting cells that can stimulate naïve T cells. However, the cells that transport antigens, tumor cell antigens in particular, have not been clearly identified. In this study we have analyzed what types of cells transport tumor cell antigens to secondary lymphoid tissues. We show that neutrophils, monocytes and macrophages but not DCs engulf X-irradiated P388 leukemic cells after their injection into the peritoneal cavity, and that neutrophils and monocytes but not macrophages migrate to the parathymic lymph nodes (pLN), the blood, and then the spleen. The monocytes in the pLN comprise Gr-1⁻ and Gr-1⁺ ones, and some of these cells express CD11c. Overall, this study demonstrates that neutrophils and monocytes transport tumor cell antigens from the peritoneal cavity to secondary lymphoid tissues.     

Characterization of trans-immortalized hepatic cell lines established from transgenic mice.

Hepato-specific regulatory (promoter/enhancer) DNA sequences were used for targeting the expression of onc genes, such as murine c-myc and Simian Virus 40 T Antigen, to hepatocytes of transgenic mice which subsequently developed hepatocellular carcinomas after a variable period of time (depending on the type of onc gene employed). Several trans-immortalized cell lines were established and compared with respect to the expression of adult hepatic markers and response to growth factors. Despite the morphological differences observed between trans-hepatomas, owing to the expression of the two different onc genes, all tumor-derived cell lines behaved in a comparable fashion during long-term culture displaying an adult hepatic phenotype for at least 40 passages. They differed, however, in response to epidermal growth factor. When the gene coding for human alpha 1-antitrypsin was placed under the control of the same hepato-specific promoter/enhancer, high levels of the human recombinant protein could be harvested from the supernatants of trans-hepatoma-derived cell lines.     

Kinetics and characterization of cellular responses in the peritoneal cavity of mice infected with Taenia crassiceps.

Changes in the leukocyte population of the peritoneal cavity ensue immediately after infection with Taenia crassiceps metacestodes. Basophils and neutrophils decrease, whereas macrophages, monocytes, and lymphocytes increase to reach only modest levels by 6 wk and then diminish to nearly disappear by 15 wk when the parasite begins rapid reproduction. Eosinophils also appear early in infection, but then abate to lower levels that persist. In late infections, when the mass of cysticerci equals that of the mouse, the cysticerci grow among surprisingly few inflammatory cells. Mingling with the peritoneal inflammatory cells is a number of odd-looking cells that could correspond to the metaplasic mesothelial cells of the host or be of parasite origin. These cells are multinucleated, they aggregate in varigerated clusters, and form cystic structures in vitro; they also bind specific anti-T. crassiceps antibodies and specific T. crassiceps DNA probes in their nuclei. When the peritoneal cell exudate is reinjected intraperitoneally into naive mice, the odd-looking cells subsist for months, inducing in the host the synthesis of specific anti-T. crassiceps antibodies and immune resistance to challenge but do not reassemble into cysticerci even after 6 mo of inoculation. The early appearance and the immunogenic and antigenic properties of these odd-looking cells suggest they are important protagonists in the early host-parasite confrontation when the outcome of infection is set.     

Characterization of myelin proteolipid mRNAs in normal and jimpy mice.

A clone specific for the rat myelin proteolipid protein (PLP) was isolated from a cDNA library made in pUC18 from 17-day-old rat brain stem mRNA. This clone corresponded to the carboxyl-terminal third of the PLP-coding region. The clone was used to identify PLP-specific mRNAs in mouse brain and to establish the time course of PLP mRNA expression during mouse brain development. Three PLP-specific mRNAs were seen, approximately 1,500, 2,400, and 3,200 bases in length, of which the largest was the most abundant. During brain development, the maximal period of PLP mRNA expression was from 14 to 25 days of age, and this was a similar time course to that for myelin basic protein mRNA expression. When the jimpy mouse, an X-linked dysmyelination mutant, was studied for PLP mRNA expression, low levels of PLP mRNA were seen which were approximately 5% of wild-type levels at 20 days of age. When jimpy brain RNA was analyzed by Northern blotting, the PLP-specific mRNA was shown to be 100 to 200 bases shorter than the wild-type PLP-specific mRNA. This size difference was seen in the two major PLP mRNAs, and it did not result from a loss of polyadenylation of these mRNAs.     

Macrophage growth inhibitors derived from the murine peritoneal cavity

Summary The murine peritoneal cavity contains factors that inhibit the in vitro growth and colony formation of macrophages. The inhibition of macrophage growth is not due to cell death. In the presence of inhibitors, the growth of colony-forming macrophages is suppressed, and small clusters are formed as a result of limited proliferation. The more mature mono-nuclear phagocytes (blood monocytes and peritoneal exudate macrophages) are more sensitive to the overall inhibitory effect of the peritoneal inhibitors than the less mature bone marrow mononuclear phagocytes. Furthermore, using dialysis and Amicon ultrafiltration, at least two inhibitors with differential inhibitory effects can be demonstrated. The colony formation of bone marrow mononuclear phagocytes is suppressed mainly by a protease-resistant, small molecular weight (<1,000) dialyzable inhibitor. In contrast, peritoneal exudate macrophages are sensitive to both the small molecular weight inhibitor and a protease-sensitive, large molecular weight (>12,000), nondialyzable inhibitor. The data suggest a possible existence of a dual inhibitor control on the proliferation of mononuclear phagocytes in vivo. In addition, the in vitro cultured peritoneal exudate cells are capable of producing inhibitors that mimic the activity of the in vivo inhibitors. This investigation was supported by Grants CA 09 11(SY) and AI15563(CCS) from the National Institutes of Health, Bethesda, MD     

Characterization of proteoglycan-reactive T cell lines and hybridomas from mice with proteoglycan-induced arthritis.

Hyperimmunization with chondroitin sulfate-depleted fetal human cartilage proteoglycan (HFPG) leads to the development of peripheral arthritis and spondylitis in BALB/c mice. Chondroitin-sulfate-depleted adult human cartilage proteoglycan (HAPG) is much less effective at inducing arthritis. These observations suggest age differences in the presence of arthritogenic proteoglycan (PG) epitopes. Earlier studies from this laboratory have indicated an important role for PG-reactive T cells in the pathogenesis of this arthritis model. To investigate further the cellular immunity to PG in mice, two T cell lines, JY.A and JY.D, and two T cell hybridomas, TH5 and TH14, were isolated from mice with PG-induced arthritis and characterized. Two patterns of reactivity to PG emerged from the analysis of these T cells. One pattern, as demonstrated by the T cell line JY.D and the two T cell hybridomas, TH5 and TH14, was characterized by reactivity to HFPG, HAPG, chondroitin sulfate-depleted bovine cartilage PG, the G1 domain (hyaluronate binding region) of bovine cartilage PG and bovine link protein. The epitope(s) recognized by these T cells appear to be part of the homologous regions shared between the G1 domain and the link protein. The second pattern of reactivity, as demonstrated by the T cell line JY.A, was characterized by reactivity to HFPG but not to HAPG or the other PG Ag or bovine link protein. All the T cell lines and hybridomas had a CD4+, CD8- phenotype, possibly belonged to the TH1 subset (IL-2+, IL-4-), and were MHC class II restricted. These studies indicate that HFPG has T cell epitopes in common with HAPG (such as in the G1 domain) and different than those in HAPG. The significance of this data in terms of PG structure, changes with age, and induction of arthritis remains to be established.     

Clinical and pathological questions concerning the tumor-like giant cell granulomas of the peritoneal cavity

Seven patients with tumor-like granulomatous lesions of the peritoneal cavity were cured, except one, by correct surgical intervention removing the inflammatory hyperplastic tissues and restoring the permeability of the alimentary tract. The exact diagnosis was suggested by the existence in the personal history of the patients of one or several interventions on the peritoneal cavity (6 of 7), and was confirmed by intraoperative, sometimes repeated, microscopic examination, rendering evident a fibrogenous giant cell granulomatous process; the presence of foreign bodies, especially suture threads or crystals (the latter characterized in polarized light) is very helpful for the diagnosis of these tumor-like inflammations.     

A comparative morphometric study on the ultrastructure of adherent cells in long-term bone marrow culture from normal and congenitally anemic mice

The relationship between structure and function of bone marrow stromal tissue in adherent layers of long-term bone marrow cultures (LTBMCs) from normal and congenital anemic mice (C57BL, Sl/Sld, Sl+/Sl+, W/Wv, and W+/W+) was investigated. Many previously reported features were confirmed. However, in LTBMC from all strains of mice examined, isolated cilia with the axonemal structure of a 9 + O pattern with obvious dynein arms were observed in the blanket cells. The frequency of cilia was approximately 2%-5% of total number of profiles of blanket cells examined. Crystalloid inclusions (CI) were observed in cultured macrophages similar to those reported in vivo in all strains of murine LTBMC. The CI could be classified into four types according to their structure in the same way as in vivo (type A to type D), with a predominance of type A in the cultures. Viral particles were also apparent in adherent cells of all strains (except W/Wv and W+/W+), which were compatible with a type C retrovirus. Gap junctions occurred regularly between the adherent cells of LTBMC, particularly between blanket cells and preadipocytes. The most frequent appearance of gap junctions was found in Sl/Sld cultures. The phenomena of normal and abnormal hematopoiesis appear to be accurately reproduced in culture, thus retaining the same relationship between function and structure as occurs in vivo. The surface of isolated cilia of blanket cells, CI of macrophages, viral particles among adherent cells, and gap junctions between blanket cells and preadipocytes is discussed.