过继转输的父系抗原耐受T细胞诱导受体母-胎免疫耐受的机制研究

To study the effect of adoptive transfer of paternal antigen-tolerant T cells on recipient reactive T cells, CBA/JxDBA/2 mating was recruited as an abortion-prone model, and CBA/JxBALB/c mating as a successful pregnancy model. The abortion-prone CBA/J females mated with DBA/2 males were injected intraperitoneally with rat anti-mouse CD80 and CD86 mAb or rat isotype IgG at day 4 after gestation (time of implantation). The purified T cells were obtained from spleen of the pregnant CBA/J mice using magnetic beads at day 9 after gestation and labeled with CFSE in vitro. The CFSE-labeled T cells were intravenously injected into other CBA/J females mated with DBA/2 males at day 4 after gestation. The proliferation of recipient splenocytes in response to DBA/2 stimulator cells was evaluated at day 9 after gestation in vitro, and the expressions of intracellular cytokines and costimulatory molecules in CFSE +/- T cells were analyzed by flow cytometry. The results showed that adoptive transfer of either paternal antigen-tolerant T cells or T cells from BALB/c-mated CBA/J mice significantly suppressed the proliferation of recipient splenocytes in response to DBA/2 stimulator cells and resulted in lower frequency of cells positive for IL-2, IFN-gamma, CD28 and higher frequency of IL-10,CTLA-4-producing cells in both CFSE+ CD3+ population and CFSE- CD3+ population compared with adoptive transfer of T cells from isotype IgG-treated CBA/J mice, whereas the frequency of IL-4-producing cells did not appear significant change. Our findings suggest that paternal antigen-tolerant T cells transferred in recipient not only function as antigen-specific suppresser cells but also disable the recipient reactive T cells, which co-suppresses maternal rejection to the allogeneic fetus, thus resulting in the decrease of the embryo resorption rate of the abortion-prone mice to that of the normal pregnancy mice.     

The effect of allogeneic lymphocytes on colony formation during culturing of hematopoietic precursor cells in the peritoneal cavity

The number of colonies formed in the peritoneal cavity (on the artificial underlayer made of peritoneal cells) and in the spleen of lethally irradiated recipients, (CBA X X C57BL) F1 mice, after the intraperitoneal injection of marrow cells depends on the cell donor's genotype: syngeneic cells and cells from mice of the parent strain CBA form fewer colonies in the peritoneal cavity than in the spleen, while cells from C57BL mice produce the reverse distribution of colonies between the peritoneal cavity and the spleen. Allogenic lymphocytes, when transplanted simultaneously with hematopoietic cells, suppress colony formation in the peritoneal cavity from day 2 of cultivation and eliminate the already developed foci of hematopoiesis by day 5.     

Nasal Flt3 ligand cDNA elicits CD11c+CD8+ dendritic cells for enhanced mucosal immunity

The embryo expresses paternal Ags foreign to the mother and therefore has been viewed as an allograft. It has been shown that anergic T cells generated by blocking of the CD28/B7 costimulatory pathway with anti B7-1 and anti B7-2 mAbs can be transferred as suppresser cells to prevent allograft rejection. Little is known, however, about the in vivo function of anti-B7-treated T cells after their transfer into abortion-prone mice in the maintenance of materno-fetal tolerance. In the present study, abortion-prone CBA/J females mated with DBA/2 males were administered anti-B7-1 and anti-B7-2 mAbs on day 4 of gestation (murine implantation window). The anti-B7-treated T cells subsequently were adoptively transferred into abortion-prone CBA/J mice. We demonstrated that costimulation blockade with anti-B7 mAbs at the time of implantation resulted in altered allogeneic T cell response and overcame increased maternal rejection to the fetus in the CBA/JxDBA/2 system. The transferred anti-B7-treated T cells appeared to be regulatory, decreasing responsiveness and generating clonal deviation in maternal recipient T cells. The transferred CFSE-labeled T cells were found to reside in the spleen and uterine draining lymph nodes, and a few were localized to the materno-fetal interface of the maternal recipient. Our findings suggest that the anti-B7-treated T cells not only function as potent suppresser cells, but also exert an immunoregulatory effect on the maternal recipient T cells, which cosuppresses maternal rejection to the fetus. This procedure might be considered potentially useful for fetal survival when used as an immunotherapy for human recurrent spontaneous abortion.     

Model system for study of cell interactions and mechanisms of immune response to T-independent antigens of type 2 in vitro

Cellular mechanisms of immune response to type 2 T-independent antigens (TI-2 antigens) are not fully elucidated up till now. In vitro system is the most convenient model for such studies. However, in vitro model requires relatively high cell density in the cultures. It hampers the study of minor lymphocyte subsets like CD5+ B-1 splenocytes, which play the main role in the immune response to TI-2 antigens. The use of cell mixtures of normal and immunodeficient congenic animals may help to resolve this problem. In this work, immune responses to TI-antigens of type 1 (TI-1 antigens) and to TI-2 antigens in vitro were studied in the mixtures of cells of normal (CBA) and congenic xid-mice (CBA/N). CBA/N mice lack CD5+ B-1 cells and do not respond to TI-2 antigens. Therefore, their splenocytes can be used as “filler” cells to create the optimal cell density in the cell cultures. Spleen and peritoneal cells of CBA mice and B-1 and B-2 lymphocytes isolated from peritoneum and spleen, respectively, were cultured in different proportions with CBA/N splenocytes with or without antigens. LPS and polyvinylpyrrolidone (PVP) were used as TI-1 and TI-2-antigens, respectively. Antibody- and immunoglobulin-forming cells (AFC and IFC, respectively) were determined by the ELISPOT method on the 4th day of cultivation. It was shown that CBA and CBA/N cells in mixed cell cultures retained their functional activity. Splenocytes of CBA mice responded to both TI-antigens. Splenocytes of CBA/N mice responded to TI-1 antigen (LPS) only. It means that in vitro B-1 cells play the main role in the immune response to TI-2 antigens, as they do in vivo. Thus, the developed model system can be used to study cellular mechanisms of immune response to TI-1 and TI-2 antigens in vitro.     

Evidence for Generation of Suppressor T Cells in Mycobacterium lepraemurium-Infected CBA/J Mice,a Mouse Strain Highly Susceptible to the Infection

Susceptibility to Mycobacterium lepraemurium (MLM) infection markedly differed between two mouse strains, CBA/J and C57BL/6. CBA/J mice showed high susceptibility to MLM infection and developed either very weak or no delayed-type hypersensitivity (DTH) to MLM antigen after the injection of MLM. In contrast, C57BL/6 mice, which were resistant to MLM infection, showed significant DTH reaction to MLM antigen after the injection. Treatment of CBA/J mice with cyclophosphamide (Cy) conferred significant resistance to MLM infection on the CBA/J mice, and the treated mice developed a strong anti-MLM DTH response after the MLM injection. When spleen cells from MLM-infected CBA/J mice were transferred to Cy-treated and MLM-infected syngeneic mice, the anti-MLM DTH reaction of the recipient mice was suppressed. Treatment of the spleen cells to be transferred with anti-Thy-1.2 antibody or anti-I-J^k antiserum plus complement abrogated the suppressive activity. Thus, it is suggested that the high susceptibility of CBA/J mice to MLM infection is due to the generation of Cy-sensitive, I-J^k-positive suppressor T cells after infection with MLM.     

Induction of antitumor L3T4-positive T cells by OK-432 at tumor sites in mice

We have previously reported the development of antitumor effector cells by day 12 after tumor implantation using a murine malignant ascites model with BAMC-1 tumor, which could be cured completely by five consecutive i.p. injections of OK-432 starting on day 2. In contrast, the OK-432 treatment with the same protocol failed to cure the tumor-bearing athymic mice, though it could suppress tumor growth temporarily. The results suggest that T cells may play a critical role in achieving a therapeutic effect. The present study was designed to clarify the nature of the antitumor effector cells induced by OK-432 in euthymic mice. The number of tumor cells in the pertioneal cavity of OK-432-treated euthymic mice increased gradually up to day 12 and dropped suddenly on day 14, while in the athymic mice the tumor cells transiently decreased in the first 7 days then started to expand drastically on day 8. The timing of the appearance of the effector cells was examined by adoptive-transfer experiments. The peritoneal exudate cells (PEC) obtained from BAMC-1 bearing euthymic mice on various days during the treatments with OK-432 were passively transferred intraperitoneally on the respective days (synchronous transfer) or on day 7 (convergent transfer) to BAMC-1-bearing athymic mice, which were treated similarly with OK-432. More than 85% of the recipient athymic mice survived when an adoptive transfer was made on and after day 7. These results indicated that the effector cells developed before day 8 in euthymic mice. The effector cells detectable on day 7 in the PEC represent plastic- or nylon-wool-column-nonadherent cells, which could cure the tumor-bearing athymic mice. Furthermore, the effector cells were destroyed when the nylon-wool-column-nonadherent cells were treated with an anti-L3T4 antibody and complement whereas the same treatment with anti-Lyt2 antibody had no effect. These L3T4⁺ cells did not possess asialo-GM1 antigen. Although the exact mechanism of action of the effector cells is yet to be clarified, the induction of human equivalents of this type of effector cell would be a good parameter indicative of clinical effects induced by OK-432 or other biological response modifiers in an individual cancer patient.     

I-Ad antigen expression of pyran copolymer-induced peritoneal cells in tumor vaccine-primed mice and its association with the host antitumor response

Summary Mice inoculated IP with L1210 murine leukemia vaccine and subsequently with pyran copolymer-induced macrophages (pyran-M) lived for a prolonged time after live L1210 inoculation IP. Pyran-M as tentatively identified by anti-AcM.1 monoclonal antibody expressed I-A^d antigen in tumor vaccine-primed recipient mice and contributed to maintaining I-A^d antigen positive (I-A^d+) macrophages at high cell density in the peritoneal cavity of recipient mice. The relevance of these I-A^d+ cells to the host antitumor response was examined by experiments in which I-A^d+ cell density in the peritoneal cavity and host antitumor response behaved in a parallel fashion. Human interferon-A/D, an agent selectively inhibiting Ia antigen expression, and silica, a general antimacrophage agent, strongly suppressed I-A^d antigen expression of peritoneal mcrophages of tumor vaccine-primed and pyran-M-inoculated mice and, consistently with this, the antitumor response was nullified in these mice. Tumor vaccine-primed mice inoculated with sodium caseinate or thioglycollate-induced peritoneal cells did not survive L1210 inoculation and, in these mice, I-A^d+ peritoneal macrophages were suppressed in number as compared with those of tumor vaccine-primed and pyran-M-inoculated mice. These results warrant further study on the contribution of I-A^d+ macrophages to pyran copolymer-induced augmentation of the antitumor response in tumor vaccine-primed mice.     

Antitumor effect of the streptococcal preparation OK-432 in a murine model of ovarian cancer

Summary The antitumor effects of the streptococcal preparation OK-432 were analyzed in a murine ovarian teratocarcinoma (MOT) model. Administration of OK-432 i.p. prevented tumor outgrowth in 75% of mice challenged with 10³ MOT cells i.p. 24 h previously. Treatment was less successful in mice challenged with 10⁴ or 10⁵ cells, preventing tumor growth in 25% of the former and only 5% of the latter group. Tumor-challenged mice cured by injections of OK-432 were not rendered resistant to a subsequent challenge with 10³ MOT cells 75 days after initial treatment. Only the i.p. route of administration was effective as i.v. OK-432 did not prolong survival of tumor-challenged mice. An antitumor response was detected as early as 24 h after i.p. treatment. This correlated temporally with an influx of neutrophils into the peritoneal cavity. Peritoneal cells obtained between 6 and 24 h after treatment were capable of lysing MOT targets in vitro. A single cell cytotoxicity assay demonstrated that peritoneal neutrophils, elicited by i.p. injection of OK-432, could bind to and lyse MOT targets. These data indicate that OK-432 is effective against small tumor cell inocula in this murine model of ovarian cancer and, furthermore, that the neutrophilic response into the peritoneal cavity plays a role in tumor rejection.     

Activity and Properties of Macrophage Migration Inhibitory Factor produced by Mixed Lymphocyte Cultures

THE macrophage migration test is an in vitro demonstration of delayed hypersensitivity. Supernatant fluids of sensitive lymphocytes cultured for 24 h in the presence of specific antigen contain migration inhibitory factor (MIF) that arrests the migration of macrophages of unsensitized animals in vitro¹,². In vivo, it induces delayed skin reactions³. The use of the macrophage migration test, based on differences of transplantation antigens in donor and recipient, to show histocompatibility has been suggested⁴. The test was also recommended as an indicator of immunological reactivity after organ transplantation, to demonstrate impending rejection⁵. It can demonstrate homograft sensitivity, for migration of peritoneal exudate cells (containing lymphocytes and macrophages) of CBA mice previously sensitized by grafts from A/Jax donors was inhibited when they were mixed with peritoneal exudate cells of the donor strain. However, histocompatibility was not demonstrated, for mixtures of peritoneal exudate cells of ungrafted CBA mice and A/Jax mice migrated regularly during the 24 h test⁶.     

Enhanced accumulation of inflammatory neutrophils and macrophages mediated by transfer of T cells from mice immunized with Listeria monocytogenes

We have previously shown that listeria-immunized mice recruit more inflammatory neutrophils and macrophages to the peritoneal cavity after i.p. injection of a sterile irritant than do nonimmune mice. Because the inflammatory phagocytes that were obtained from listeria-immune and nonimmune mice did not differ in their ability to kill Listeria monocytogenes in vitro, this suggested that the rapid recruitment of listericidal inflammatory neutrophils and macrophages may be critically important for resistance to listeriosis. In this study we demonstrate that the transfer of listeria-immune T cells, which enhances recipient resistance to listeriosis, also increases the ability of recipients to mobilize inflammatory neutrophils and macrophages to the peritoneal cavity after the i.p. injection of dead listeria. The transfer of enhanced inflammatory responsiveness was blocked by pretreatment of the transferred cells with anti-Thy-1.2 plus complement, and the magnitude of the inflammatory cell accumulation was dependent on the number of listeria-immune T cells that were injected. Inflammatory neutrophils and macrophages that were obtained from the mice after the transfer of listeria-immune or nonimmune T cells (plus dead listeria) did not differ in their ability to kill L. monocytogenes in vitro. These data suggest that the elicitation of an inflammatory response may be an important event in T cell-mediated resistance to listeriosis.     

Antitumor and immunologic effects of a pyridine-extracted fraction of Propionibacterium acnes

Summary The antitumor and immunological effects of a pyridine extractable fraction of Propionibacterium acnes were tested in a murine ovarian teratocarcinoma (MOT) model. Previous studies have demonstrated that tumor rejection in this model depends upon sequential activation of tumoricidal neutrophils (PMNs) followed by cytostatic macrophages. The pyridine extract significantly prolonged the survival of mice challenged with 10³ or 10⁴ MOT cells but had little impact on a 10⁵ tumor inoculum. In vivo cytoreduction occurred within the first 24 h following IP treatment which correlated temporally with the influx of tumoricidal PMNs into the peritoneal cavity. Immunotherapy failure in mice challenged with 10⁵ MOT cells occurred between 48 and 72 h after treatment when macrophage chemotaxis into the peritoneal cavity was initiated. Although injection of unfractionated bacteria activated MOT-cytostatic macrophages, the pyridine extract was deficient in this regard. Intraperitoneal injection of the pyridine extract resulted in an early (day +1) depression and late (day +5) enhancement of peritoneal NK cytotoxicity. These data suggest that the retention of neutrophil-activating moieties in the pyridine extract are sufficient for antitumor effects against low tumor inocula while the depletion of macrophage-activating determinants results in diminished effects against larger tumor cell challenges.     

Delayed clearance of filarial infection and enhanced Th1 immunity due to modulation of macrophage APC functions in xid mice.

Bruton's tyrosine kinase (Btk) mutant CBA/N mice show delayed clearance of injected microfilaria (mf) compared with wild-type CBA/J mice. Anti-mf T cells from CBA/N mice make relatively more IFN-gamma than those from CBA/J mice. The anti-mf T cell proliferative responses are also greater in CBA/N mice. This CBA/N immune phenotype is not restricted to filarial Ags, because immunization with pure proteins also yields T cell responses of greater proliferative magnitude skewed away from Th2 cytokines in CBA/N compared with CBA/J mice. The increased magnitude of CBA/N T cell proliferative responses is reflected in increases in both precursor frequencies and clonal burst sizes of responding Ag-specific T cells, and is independent of the source of re-stimulating APCs. Transfer of CBA/J peritoneal resident cells (PRCs) into CBA/N mice before pure protein immunization leads to a wild-type immune phenotype in the recipient CBA/N mice, with a reduction in the proliferative response and a relative decrease in the IFN-gamma produced. When wild-type PRC subpopulations are similarly transferred, the wild-type immune phenotype is transferred by macrophages rather than by B cells. Transfer of wild-type PRCs into CBA/N mice before injection of mf also causes similar changes in the anti-mf T cell responses and enhances the clearance of mf. Thus, Btk is involved in critical macrophage APC functions regulating priming of T cells, and can modulate these responses in pathophysiologically relevant fashion in vivo.     

The role of host lymphocytes and host macrophages in antitumor reactions after injection of sensitized lymphocytes and tumor target cells into naive mice

Summary DBA/2 mice were immunized i.p. against syngeneic SL2 lymphosarcoma cells. At various days after the last immunization peritoneal and spleen lymphocytes were collected. The lymphocyte suspensions were enriched for T-cells by nylon wool filtration.The peritoneal T-cells from immunized mice (a) expressed direct specific antitumor cytotoxicity in vitro, (b) induced macrophage cytotoxicity in vitro, and (c) exerted tumor neutralization measured in a Winn-type assay. Spleen T-cells from these immunized mice (a) expressed no direct specific antitumor cytotoxicity in vitro, (b) only induced moderate macrophage cytotoxicity in vitro, but (c) exerted tumor neutralization in a Winn assay.For effective tumor neutralization in vivo effector target cell ratios of 1000:1 were required. When the effector/target ratio of 1000:1 was maintained but the absolute numbers of effector and target cells were lowered from 10⁶ to 10⁵ lymphocytes and 10³ to 10² target cells respectively, no tumor neutralization was obtained.The major effect of the sensitized-transferred T-lymphocytes seemed to be the induction of cytotoxic macrophages in the (naive) recipient mice, as the peritoneal macrophages collected from the recipient mice 7 days after i.p. injection of a mixture of sensitized T-cells and tumor cells were cytotoxic. Purified peritoneal T-lymphocytes collected from these recipient mice were able to induce macrophage cytotoxicity in vitro but expressed no cytotoxic T-cell activity.In conclusion, our results show that in the tumor system used, tumor neutralization after transfer of sensitized lymphocytes is not dependent on the presence of cytotoxic T-lymphocytes. Lymphocytes with the strongest potency to render macrophages cytotoxic (in vitro and in vivo) also induce the best tumor neutralization in vivo, suggesting an important role for host macrophages as antitumor effector cells.     

The role of the peritoneal cavity in successful treatment of a murine lymphoma with chemotherapy and non-specific immunostimulation

Summary The influence of the route of administration and treatment schedule of a yeast immunoadjuvant, Candida albicans (CA) on the degree of success achieved with an immunochemotherapy regimen in a virus-induced murine lymphoma has been evaluated. To this end, histocompatible CD2F1 mice received IP or IV inoculations of LSTRA lymphoma cells and were subjected to various treatments with inactivated CA and bis, 1, chloroethyl nitrosourea (BCNU).The results showed that CA may significantly increase the antitumor efficiency of BCNU when (a) the tumor is inoculated IP and not IV; (b) CA is administered before (on day –14) and after (on days +1 and/or day +8) LSTRA challenge; (c) CA is given IP as a post-tumor treatment.To ascertain whether the immunoadjuvant effect was anatomically restricted to the peritoneal cavity (PC), spreading of IP injected lymphoma was studied by means of LSTRA cells labeled with 3–5iodo-deoxyuridine ¹²⁵I (¹²⁵IUdR) and tumor bioassay in spleen, lung, kidney, liver, and PC of recipient mice. The results showed that IP tumor challenge led to early (1 h) generalized neoplasia in both untreated and CA-pretreated hosts.Therefore, the combined antitumor effects of chemotherapy and CA are not restricted to the PC but rather the result of systemic immunity. In conclusion, in our system the PC seems to be a preferential site for eliciting generalized antilymphoma host responses markedly amplified by selected schedules of immunoadjuvant administration.     

Peritoneal cavity B cells are precursors of splenic IgM natural antibody-producing cells

Peritoneal cavity B-1 cells are believed to produce IgM natural Abs. We have used alpha1,3-galactosyltransferase-deficient (GalT(-/-)) mice, which, like humans, produce IgM natural Abs against the carbohydrate epitope Galalpha1,3Gal (Gal), to demonstrate that peritoneal cavity B-1b cells with anti-Gal receptors produce anti-Gal IgM Abs only after LPS stimulation. Likewise, peritoneal cavity cells of GalT(-/-) and wild-type mice do not produce IgM Abs of other specificities without LPS stimulation. Development of Ab-secreting capacity is associated with loss of CD11b/CD18 (Mac-1) expression. In contrast, there are large numbers of cells producing anti-Gal and other IgM Abs in fresh splenocyte preparations from GalT(-/-) and (for non-Gal specificities) wild-type mice. These cells are Mac-1(-) but otherwise B-1b-like in their phenotype. We therefore hypothesized a pathway wherein peritoneal cavity B cells migrate into the spleen after activation in vivo and lose Mac-1 expression to become IgM Ab-producing cells. Consistent with this possibility, splenectomy reduced anti-Gal Ab production after immunization of GalT(-/-) mice with Gal-positive rabbit RBC. Furthermore, splenectomized B6 GalT(-/-), Ig micro -chain mutant ( micro (-/-)) (both Gal- and B cell-deficient) mice produced less anti-Gal IgM than nonsplenectomized controls after adoptive transfer of peritoneal cavity cells from B6 GalT(-/-) mice. When sorted GalT(-/-) Mac-1(+) peritoneal cavity B cells were adoptively transferred to B6 GalT(-/-), micro (-/-) mice, IgM Abs including anti-Gal appeared, and IgM-producing and Mac1(-) B cells were present in the spleen 5 wk after transfer. These findings demonstrate that peritoneal cavity Mac-1(+) B-1 cells are precursors of Mac-1(-) splenic IgM Ab-secreting cells.     

CD40 ligation restores cytolytic T lymphocyte response and eliminates fibrosarcoma in the peritoneum of mice lacking CD4+ T cells

Absence of CD4⁺ T cell help has been suggested as a mechanism for failed anti-tumor cytotoxic T lymphocytes (CTL) response. We examined the requirement for CD4⁺ T cells to eliminate an immunogenic murine fibrosarcoma (6132A) inoculated into the peritoneal cavity. Immunocompetent C3H mice eliminated both single and repeat intraperitoneal (IP) inoculums, and developed high frequency of 6132A-specific interferon-γ (IFNγ)-producing CTL in the peritoneal cavity. Adoptive transfer of peritoneal exudate cells (PEC) isolated from control mice, protected SCID mice from challenge with 6132A. In contrast, CD4 depleted mice had diminished ability to eliminate tumor and succumbed to repeat IP challenges. Mice depleted of CD4⁺ T cells lacked tumor-specific IFNγ producing CTL in the peritoneal cavity. Adoptive transfer of PEC from CD4 depleted mice failed to protect SCID mice from 6132A. However, splenocytes isolated from same CD4 depleted mice prevented tumor growth in SCID mice, suggesting that 6132A-specific CTL response was generated, but was not sustained in the peritoneum. Treating CD4 depleted mice with agonist anti-CD40 antibody, starting on days 3 or 8 after initiating tumor challenge, led to persistence of 6132A-specific IFNγ producing CTL in the peritoneum, and eliminated 6132A tumor. The findings suggest that CTL can be activated in the absence of CD4⁺ T cells, but CD4⁺ T cells are required for a persistent CTL response at the tumor site. Exogenous stimulation through CD40 can restore tumor-specific CTL activity to the peritoneum and promote tumor clearance in the absence of CD4⁺ T cells.Supported in part by grants from Children’s Hospital of Wisconsin Foundation, Society of University Surgeons Foundation, Florence and Marshall Schwid Foundation, Elsa Pardee Foundation, Kathy Duffy Fogarty Fund of the Greater Milwaukee Foundation (JS) and NIH grant RO1-CA-37156 (HS); Andrew Lodge and Ping Yu have contributed equally to this work.     

Binding of low-density lipoproteins from chicken egg yolk to mouse anti-phospholipid B cells which include B cells against bromelain-treated mouse erythrocytes

Low-density lipoproteins from chicken egg yolk (EyLDL), which are reactive with mouse antibodies against bromelain-treated mouse erythrocytes (BrMRBC), were conjugated with fluorescein isothiocyanate (FITC). FITC-EyLDL could bind specifically to mouse anti-phospholipid B cells, which comprised all the BrMRBC-rosette-forming cells and anti-BrMRBC lipopolysaccharide-reactive B cells, C3H/He mice at 12 weeks of age had, approximately, 7 x 10(5) EyLDL-binding cells in the peritoneal cavity, 3 x 10(5) EyLDL-binding cells in the pleural cavity, and 3 x 10(5) EyLDL-binding cells in the spleen. In ontogeny, the numbers of EyLDL-binding cells in the peritoneal cavity expanded greatly by 4 weeks. Other normal strains of mice and C3H/HeJ mice at 12 weeks of age had 4-7 x 10(5) EyLDL-binding cells in the peritoneal cavity; the numbers were large (19 x 10(5] in NZB mice, rather small (2 x 10(5] in MRL/lpr mice, and very small (0.1 x 10(5] in CBA/N mice. In some of various strains of mice at 12 months of age, more than 20% of peritoneal cells were EyLDL-binding cells; in particular, all of five older NZB mice examined had more than 10(7) EyLDL-binding cells in the peritoneal cavity.     

CD5+ peritoneal B cells express high levels of membrane, but not secretory, C mu mRNA.

We used in situ hybridization to study Ig mRNA levels in murine peritoneal and splenic B cells. Ig mRNA production fell into three distinct groups: low, intermediate, and high. Splenic B cells primarily exhibited low levels characteristic of resting B cells or high Ig mRNA levels characteristic of plasma cells. In contrast, a significant fraction of peritoneal B cells exhibited intermediate Ig mRNA levels. Intermediate Ig mRNA was T cell dependent in that congenic nu/nu mice had far fewer peritoneal cells expressing the intermediate Ig message than their wild type counterparts. CD5+ CD11b+ IgMbright+ peritoneal B cells were found to be mainly responsible for the production of intermediate Ig mRNA levels. The peritoneal CD5- CD11b+ IgMbright+ "sister" B cell subpopulation contained a lower percentage of intermediate Ig mRNA-producing B cells. CD5-CD11b-IgMdull+ "conventional" B cells produced negligible levels of Ig mRNA, comparable to those of unfractionated splenic B cells. Northern analysis showed that the majority of Ig mRNA expressed in the peritoneum is of the membrane rather than the secreted form. Consistent with that result, in short-term culture, peritoneal cells showed markedly less Ig secretion than did spleen cells. These studies describe novel Ig mRNA expression by peritoneal B cells and emphasize that within the peritoneal cavity, B cells do not tend to become antibody-secreting cells.     

A distinct subset of podoplanin (gp38) expressing F4/80+ macrophages mediate phagocytosis and are induced following zymosan peritonitis

Macrophages are important tissue resident cells that regulate the dynamics of inflammation. However, they are strikingly heterogeneous. During studies looking at podoplanin (gp38) expression on stromal cells in the murine spleen and peritoneal cavity we unexpectedly discovered that podoplanin was expressed on a subset of F4/80⁺ macrophages; a subset which we have termed fibroblastic macrophages (FM). These cells function as phagocytes in vitro as measured by bead mediated phagocytosis assays. FM also exist at high frequency in the peritoneal cavity and in zymosan induced peritonitis in vivo. These FM represent a unique subgroup of F4/80⁺ macrophages and their presence in the inflamed peritoneum suggests that they play a role in zymosan induced peritonitis.     

The in vitro bactericidal activity of peritoneal and spleen cells from Listeria-resistant and -susceptible mouse strains

Two days after Listeria-resistant (LrR) C57BL/10 mice were infected intraperitoneally with Listeria, their peritoneal macrophages demonstrated enhanced bactericidal activity beyond that seen in susceptible (LrS) BALB/c or CBA mice. Intravenous infection had no effect on peritoneal cell activity. The induction, but not expression, of the enhanced activity was radiosensitive. There was no significant difference between the strains with respect to the number of cells or cellular composition of the exudates. No difference in the in vitro chemotactic response of cells from the two strains could be demonstrated. Therefore there seems to be recruitment to the infected peritoneal cavity of C57BL/10 mice of young, efficiently bactericidal monocytes/macrophages. On the other hand, spleen cell bactericidal activity was intrinsically superior in C57BL/10 mice compared with BALB/c mice, possibly because, as a haemopoietic organ, the C57BL/10 spleen already contains high numbers of these efficient monocytes.