Cytotoxic effect of endodontic bacteria on periapical fibroblasts

This study was conducted to investigate the effects of sonicated bacterial extracts (SBEs) from anaerobic Gram-negative bacteria on periapical fibroblast obtained from the apical portion of human periodontal ligaments. Porphyromonas endodontalis, Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum were chosen from among the endodontic bacteria isolated from root canals having a periapical lesion and compared in terms of their cytotoxicity. The purpose of this study was to examine which bacteria are involved in the development of periapical inflammation. The anaerobes were cultured under strict anaerobic conditions, and the bacterial cells were then harvested by centrifugation after incubation. The concentrated cell suspensions were sonicated and subsequently centrifuged. An SBE was made of each of the filtered supernatants. Each SBE was added to cultures of periapical fibroblasts. The cell growth and proliferation were measured by the MTT method after 3, 5, and 7 days. The SBEs from P. endodontalis, P. gingivalis, and F. nucleatum inhibited the growth of the fibroblasts, whereas the SBE from P. intermedia did not inhibit it. The SBEs from P. gingivalis and F. nucleatum inhibited the fibroblast growth more strongly than did the P. endodontalis, P. gingivalis, and F. nucleatum may participate in the development of periapical lesions.     

The concepts prevalence, need for treatment, and prevention of temporomandibular disorders: a suggestion for terminology

To determine the effect of pathogenic oral bacteria on interleukin 6 (IL-6) and soluble IL-6 receptor production, we measured their release by human peripheral blood mononuclear cells in vitro. Unseparated peripheral blood mononuclear cells, peripheral blood lymphocytes (monocyte depleted), pure T cells, or monocytes were cultured with Actinobacillus actinomycetemcomitans, Capnocytophaga gingivalis, Capnocytophaga ochracea, Fusobacterium nucleatum or Porphyromonas gingivalis for 24 h. Supernatants were tested for IL-6 and soluble IL-6 receptor by enzyme-linked immunosorbent assay. Only monocytes and peripheral blood mononuclear cells responded with significant IL-6 release in the presence of all bacteria tested. However, peripheral blood lymphocytes were capable of producing IL-6 when activated by phytohemagglutinin or IL-2 followed by bacteria, though substantially less than cultures containing monocytes. No bacteria tested increased soluble IL-6 receptor release over spontaneous soluble IL-6 receptor release. We conclude that monocytes release IL-6 after contact with oral pathogens; however, soluble IL-6 receptor from T cells and monocytes is constitutively produced and may modulate IL-6 actions.     

Reduced serum IgG reactivities with bacteria from dental plaque in HIV-infected persons with periodontitis

Serum samples were obtained from 44 HIV-seropositive (HIV+) and 37 HIV-seronegative (HIV-) persons that were grouped according to periodontal status. Serum IgG and IgA reactivities towards Streptococcus mutans, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis. Prevotella intermedia, Prevotella nigrescens and Fusobacterium nucleatum were measured by means of ELISA. HIV+ persons with chronic marginal periodontitis showed significantly lower IgG reactivities to the periodontal pathogens A. actinomycetemcomitans, P. gingivalis, P. intermedia and F. nucleatum as compared with their HIV- counterparts (p < 0.05). Specific serum IgA reactivities were similar in the two periodontitis groups, except for P. nigrescens where the HIV+ group with chronic marginal periodontitis had lower values than their systemically healthy counterparts (p < 0.05). The results indicate that HIV infection affects the humoral serum immune responses against bacteria in dental plaque; the depressed antibody responses may contribute to the increased susceptibility for periodontal infections in HIV-infected patients.     

Characterization of the mitogenic response of murine CD5+ and conventional B lymphocytes to lipopolysaccharide

The nature of the response of conventional and CD5+ B cells to stimulation in vitro with optimal mitogenic concentrations of LPS was examined to elucidate the contributions of these B cell subsets in polyclonal B lymphocyte responses. Stimulation of murine splenic lymphocytes with LPS resulted in an increase in total biomass, peaking at 72 h of culture. The viability of the cultures remained high (> 90%) until 48 h of culture. A combination of trypan blue and 7-aminoactinomycin D (7AAD) exclusion in conjunction with PE-anti-CD5 and FITC-anti-B220 enabled more detailed analysis of the cultures. The total number of conventional B cells, viable and non-viable, increased until 48 h of culture and then decreased when stimulated with LPS, while CD5+ B cells increased over the culture period. The numbers of conventional B cells in the control cultures decreased, but the CD5+ B cell numbers remained stable. An examination of the modes of death of the B cell subsets using 7AAD showed that unstimulated conventional B cells were apoptotic rather than degenerate but, following stimulation with LPS, apoptotic and degenerate cells were found. Apoptotic and degenerate CD5+ B cells were found in both stimulated and unstimulated cultures, but the percentage of these apoptotic and degenerate cells was increased significantly only at 72 h and 96 h of culture in stimulated cultures compared with 24 h onwards in the control cultures. Morphological analysis and gel electrophoretic studies of extracted DNA reflected these findings. It was also found that the increase in the number and percentage of non-viable cells in the cultures was not equal to the decrease in the number and percentage of viable cells. Activation of B cells was examined using expression of B7-1 (CD80) as a marker. When stimulated with LPS a greater proportion of conventional B cells expressed B7-1 after 24 h of culture than in the control cultures; however, only at 72 h and 96 h of culture was the proportion of CD5+ B cells expressing B7-1 significantly higher than in the control cultures. These results show that conventional B cells are stimulated to proliferate and to become activated by LPS and that death is apoptotic rather than degenerate or necrotic. CD5+ B cells were also shown to be stimulated by LPS; they became activated and death was delayed. The data suggest that in addition to the proliferative role, LPS acts to delay death and to activate conventional and CD5+ B cells.     

Migration and activation of antigen-specific CD8+ T cells upon in vivo stimulation with allogeneic tumor

The response of CD8+ T cells to allogeneic tumor was studied by adoptive transfer of cells from TCR transgenic 2C mice specific for Ld alloantigen. Transferred cells were monitored during the course of a response to i.p. challenge with live P815 (H-2d) using the 1B2 mAb specific for the 2C TCR. Tumor was present in the draining LN and spleen within 3 to 4 days of challenge. The first changes in 1B2+ cells occurred in the spleen on day 4; VLA-4 expression increased in an Ag-specific manner and L-selectin expression decreased in an Ag-nonspecific manner. The number of 1B2+ cells in the spleen declined over days 4 to 6. The first detectable increase in CD25 expression and blast transformation was in the peritoneal cavity beginning days 5 and 6. Clonal expansion was largely limited to this site and was maximal on day 8. As expansion occurred in the peritoneal cavity; the number of 1B2+ cells in the draining LN and spleen also increased. These cells had an activated phenotype (CD44(high), VLA-4(high)) but most did not express CD25 and were not blasts. These results suggest that initial Ag recognition in the spleen results in altered expression of adhesion receptors so that cells gain access to the peritoneal cavity where they undergo clonal expansion and differentiation. Following the response, 1B2+ cells decline in number but a memory population (CD44(high), L-selectin(high and low)) persists for long times in the spleen and LN.     

Stimulation of basal and L-DOPA-induced motor activity by glutamate antagonists in animal models of Parkinson''s disease

Peripheral T-cell antigen receptor V beta (TCRV beta) repertoire is influenced by clonal deletion both in the thymus and periphery. Developing thymocytes expressing certain TCRV beta are deleted by endogenous superantigens presented on major histocompatibility complex (MHC) molecules in the thymus. Likewise, mature T cells bearing particular TCRV beta chains can be clonally deleted by superantigens in the periphery. The efficiency with which T cells expressing particular V beta subunits are deleted differs depending upon which coreceptor is expressed. Indeed, while deletion of V beta 11+ splenic T cells in CBA/J (Mls-1, a I-E, + MTV 9+) mice is quite efficient for CD4+ spleen T cells, it is much less efficient for CD8+ splenic T cells. If the difference in the efficiency of deletion is due solely to the coreceptor expressed, then a transgene encoding CD4 should increase the efficiency with which CD8+ cells are deleted. To address this question, we have produced CD4 transgenic (TG) mice that express physiologic levels of CD4 on all thymocytes and peripheral CD8 T cells. CD4 molecules expressed on CD8+ splenic T cells were associated with P56lck tyrosine kinase, and were functional as evidenced by their ability to facilitate class II alloreactivity. Furthermore, we found that ectopic expression of TG CD4 molecules on CD8+ cells was able to affect the efficiency of deletion in response to superantigen stimulation. In particular, deletion of TCRV beta 11+ T cells was much less efficient for CD8+ than for CD4+ T-cell subpopulations in (CBA/J x B6) F1 mice. However, expression of the CD4 transgene on CD8+ splenic T cells from these mice increased the efficiency of deletion in the CD8+ V beta 11 T cells. Interestingly, this effect was not observed in a mature CD8+ thymocyte subpopulation. The results in this report demonstrate that CD4 molecules are involved in peripheral deletion of TCRV beta 11+ T cells in (CBA/J x B6) F1 mice, and that the TCRV beta repertoire can be altered by ectopic expression of CD4 on all T-lineage cells.     

Immune response to anaerobic bacteria in patients with peritonsillar cellulitis and abscess

The role of four oral organisms (Fusobacterium nucleatum. Prevotella intermedia, Porphyromonas gingivalis, and Actinobacillus actinomycetemcomitans) was investigated in 19 children with peritonsillar abscess, and 17 with peritonsillar cellulitis. Antibody titers to these organisms were measured by enzyme- linked immunosorbent assay in the patient, as well as in 32 control patients. Serum levels in the patients were determined at day 1 and 42-56 days later. Significantly higher antibody levels to F. nucleatum and P. intermedia were found in the second serum sample of patients with peritonsillar cellulitis or abscess, as compared to their first sample or the levels of antibodies in controls. A total of 136 bacterial isolates, 100 anaerobic and 36 aerobic were isolated from the 19 peritonsillar abscesses. Anaerobic bacteria were found in all abscesses, and they were mixed with aerobic bacteria in 5 (26%). F. nucleatum was recovered in 14 (74%) abscesses and P. intermedia was isolated in 13 (68%). The elevated antibody levels to F. nucleatum and P. intermedia, known oral pathogens, suggest a pathogenic role for these organisms in peritonsillar infections.     

Neuroimmune control of interleukin-6 secretion in the murine spleen. Differential beta-adrenergic effects of electrically released endogenous norepinephrine under various endotoxin conditions

In a previous study we demonstrated a superfusion technique which allows for investigation of nerve-immune cell interaction in murine spleen. We demonstrated that under septic-like conditions in the presence of bacteria and lipopolysaccharide (LPS), electrically induced inhibition of interleukin 6 (IL-6) secretion was attenuated by the beta-adrenergic antagonist propranolol. This effect was now investigated more closely under various endotoxin conditions in order to dissect effects of bacteria and endotoxin: (A) bacteria-rich conditions (without penicillin/streptomycin [P/S] and without LPS), (B) LPS-enriched conditions (with P/S and with LPS), and (C) bacteria-free conditions (with P/S and without LPS). Under bacteria-rich conditions, norepinephrine (Emax = 10(-6) M, p = 0.012) and isoproterenol (Emax = 10(-6) M, p = 0.048) concentration-dependently inhibited IL-6 secretion from murine spleen slices in contrast to bacteria-free conditions. In a bacteria-free environment the beta-adrenergic antagonist propranolol did not attenuate the electrically induced inhibition of splenic IL-6 secretion. The insertion of bacterial filters in front of the superfusion chambers to avoid direct contact between bacteria and cells increased the electrically-induced inhibition of IL-6 secretion (p = 0.0036). Added LPS did not change the electrically-induced release of norepinephrine from presynaptic nerve terminals in murine spleen. The study demonstrates two different beta-adrenergic effects on IL-6 secretion of murine spleen slices under bacteria-rich or bacteria-free conditions.     

Effect of macrophage colony-stimulating factor (M-CSF) on mouse immune responses in vivo

We examined the effects of recombinant human M-CSF (rhM-CSF) on mouse macrophages and immune responses in vivo. Intraperitoneal administration of rhM-CSF (20-500 microgram/ml) increased Mac-1+ cell numbers in the peritoneal cavity. The tumoricidal activities of the macrophages from vehicle-administered (V-M phi) and from rhM-CSF-administered (M-M phi) mice were the same as those observed in vitro. However, when activated by lipopolysaccharide (LPS), the tumoricidal activity of M-M phi was stronger than that of V-M phi. Intravenous administration of rhM-CSF (500 micrograms/gk) increased the number of spleen cells. Flow cytometric analysis showed that administration of rhM-CSF increased Mac-1+, B220+ and NK 1.1+ cell counts in the spleen. However, CD4+ and CD8+ cell numbers did not change. Concomitant increases were observed in levels of IL-4 and IL-10 in mouse serum following rhM-CSF administration, but no significant changes were observed in the serum level of IFN-gamma. In experiments involving mouse immune responses, the administration of rhM-CSF reduced the contact sensitivity (CS) reaction against picryl chloride (PC) and augmented IgE production in response to 2,4-dinitrophenyl (DNP), but did not affect the production of either IgM or IgC1. These results suggest that administration of rhM-CSF not only activates murine macrophages, but modulates antigen-specific immune responses in vivo.     

CD1 expression defines subsets of follicular and marginal zone B cells in the spleen: beta 2-microglobulin-dependent and independent forms

We have used multicolor FACS analysis, immunohistology, and functional assays to study the expression of CD1 on B cell subsets from normal and beta 2m-/- mice. Two B cell subpopulations were identified that express high levels of CD1 in normal mice: splenic marginal zone B cells (IgMhigh IgDlow CD21high CD24intermediate CD23- CD43-) and a newly identified subpopulation of follicular B cells. The latter cells are unusual, because they are IgDhigh CD23+, like follicular B cells, but express high levels of CD21 and IgM, an expression pattern that is associated with marginal zone B cells. Therefore, the high-level expression of CD1 and CD21 was found to be closely associated on splenic B cells. Immunohistology confirmed the expression of CD1 on marginal zone B cells and on clusters of B cells in splenic follicles. Both the high-level CD1 expression by these cells and the low-level CD1 expression by subpopulations of B cells in the spleen, lymph node, peritoneal cavity, and bone marrow were markedly reduced in beta 2m-/- mice. Despite this, a CD1-restricted T cell clone proliferated vigorously in response to LPS-activated spleen cells that had been obtained from both beta 2m-/- and wild-type mice. This response was inhibited by the 3C11 anti-CD1 mAb. These results show the heterogeneity of B cell subsets in their expression of the beta 2m-dependent form of CD1. They further suggest that a beta 2m-independent form of CD1 is expressed on B cells that can stimulate T cells; however, this form is not easily visualized with the anti-CD1 mAb used here.     

Preparation and characterization of an Actinomyces naeslundii aggregation factor that mediates coaggregation with Porphyromonas gingivalis

Intergeneric coaggregation is responsible for the complexity of the microbiota in human dental plaque and is believed to be important in the initial bacterial colonization of the human oral cavity. Actinomyces naeslundii, an early colonizer of the tooth surface, may enhance subsequent colonization by Porphyromonas gingivalis which is associated with adult periodontitis. The purpose of this study was to isolate and characterize the A. naeslundii aggregation factor (AnAF) that mediates coaggregation with P. gingivalis. AnAF was isolated from A. naeslundii sonic extract (SE) by gel filtration on a Sephacryl S-400HR, by hydrophobic interaction chromatography on a HiTrap Octyl Sepharose 4FF, and by ion exchange chromatography on a HiTrap Q. The specific activity increased 12-fold with a yield of 2.5%. SDS-PAGE analysis of AnAF revealed a protein band of high molecular weight in excess of 200 kDa. Carbohydrate was detected as the only material coinciding with the protein band, indicating that the AnAF was a glycoprotein. Immunoblotting analysis indicated that AnAF directly bound to P. gingivalis cells. AnAF was sensitive to sodium metaperiodate treatment but not to heat or protease treatments. These results suggest that the AnAF carbohydrate component mediated coaggregation with P. gingivalis cells. AnAF also inhibited coaggregation with other periodontal disease-associated bacteria such as Prevotella intermedia, Fusobacterium nucleatum, Capnocytophaga ochracea, but not streptococci.     

Expression of mouse CD43 in the B cell lineage of transgenic mice causes impaired immune responses to T-independent antigens

CD43 (leukosialin), a sialylated glycoprotein expressed on the surface of most hematopoietic cells, has been implicated in cell adhesion and signaling. However, its precise physiological function remains unclear. We used mouse CD43 (mCD43)-immunoglobulin enhancer-transgenic (TG) mice to study the role of mCD43 in vivo. Previous work revealed that mCD43 expression on mature B cells in these mice resulted in immunodeficiency to T-dependent (TD) antigens (Ag), possibly by impairing B-T cell interactions. In the present study we have immunized the TG mice with the T-independent (TI) Ag fluorescein-(Fl) lipopolysaccharide (LPS) (TI type 1 Ag) and Fl-Ficoll (TI type 2 Ag). Surprisingly, the mCD43-Ig enhancer expressing mice were impaired in their ability to mount humoral responses to both Fl-LPS and Fl-Ficoll, and had decreased numbers of cells responding to Ag in vivo. Flow cytometric analysis was performed on peritoneal B-1 cells, a population which often plays a major role in humoral responses to TI Ag such as bacterial Ag. This analysis revealed similar B220, IgM and CD5 expression patterns for the TG and nontransgenic (NTG) B-1 cells. In addition, purified peritoneal B-1 cells from TG and NTG mice were able to respond to LPS. Stimulation of splenic B cells in vitro with Fl-LPS and Fl-Ficoll revealed that, in contrast to NTG B cell responses, TG B cell responses could not be enhanced by co-culture with T cells. However, soluble T cell factor enhancement of the TG B cell responses was normal. These data suggest that the mCD43 expression on B cells may inhibit cell interactions that are important for enhanced TI Ag responses. The anti-adhesive forces of mucins in general may thus be critical in regulating both TD and TI humoral responses.     

Administration of silica sensitizes lipopolysaccharide responsiveness of murine macrophages but inhibits T and B cell priming by inhibition of antigen presenting function

Macrophages play a key role in natural host defense against infection by a variety of pathogens. In addition, macrophages initiate the development of acquired immunity via antigen processing and presentation. The role of macrophages in resistance to pathogens, the development of autoimmune diseases and the induction of acquired immunity has been studied by treatment of rodents with reagents which are cytotoxic. We have studied the effects of one such reagent, silica, on the function of spleen macrophages and peritoneal exudate cells (PEC). Intraperitoneal administration of silica caused the accumulation of spleen macrophages and neutrophils, reduction in the number of B cells and had a modest effect on T cell abundance. The percentage of CD11b+ PEC was not affected by silica treatment but total PEC recovery was diminished 5-8 fold. Silica treatment did not cause release of TNF-alpha or IL-1-beta but, when stimulated with lipopolysaccharide (LPS) in vitro after silica treatment, PEC or spleen macrophages produced elevated levels of both cytokines compared to controls. In contrast, release of IL-12 from non-LPS treated PEC was stimulated 4-5 fold by silica treatment. In addition, sensitivity to LPS toxicity in vivo was significantly enhanced by silica. The ability of macrophages to present antigen to a T cell clone in vitro was found to be dramatically inhibited by silica treatment, as was the ability to prime antigen-specific T cells and B cells by antigen injection. Collectively these data demonstrate that silica treatment enhances macrophage sensitivity to LPS exposure but inhibits antigen processing and presentation.     

Immune response-mediated protection of adult but not neonatal mice from neuron-restricted measles virus infection and central nervous system disease

From the biobreeding-diabetic prone (BB-DP) rat, an animal model for endocrine autoimmunity, phenotype and function of splenic dendritic cells (DC) were studied. Furthermore, the suppressive effect of peritoneal macrophages (pMphi) from the BB-DP rat in the MLR was investigated. Lower numbers of splenic DC were isolated from BB-DP rats than from control Wistar rats. In the preautoimmune phase, DC of the BB-DP rat had a lower surface MHC class II expression (and in preliminary data, a lower CD80 expression), ingested more bacteria, and had a lower stimulatory potency in the syngeneic (syn)MLR as compared with control DC. During disease development, the MHC class II expression further decreased, and a low stimulatory activity became evident in the allogeneic (allo)MLR. With regard to the expansion of suppressor/regulatory T cells, a lower percentage of RT6+ T cells but higher percentages of CD45RClow T cells were induced by BB-DP DC in synMLR, but not in alloMLR. An increase in the CD4/CD8 T cell ratio was observed in both the syn- and alloMLR due to a relative weak expansion of CD8+ T cells with DC of the BB-DP rat. Resident pMphi isolated from BB-DP or Wistar rats were equally effective in suppressing the DC-driven synMLR. In conclusion, splenic DC from the BB-DP rat have a lower accessory cell function already at young age, before the development of disease, and expanded different subsets of effector/suppressor T cells in vitro as compared with those from Wistar rats. The dysfunction of DC from BB-DP rats is likely to be caused by their relative immaturity as indicated by their low class II and costimulatory molecule expression and relatively high phagocytic activity.     

Ecologic studies of rodent reservoirs: their relevance for human health

To gain insight into the immunomodulatory effects of vitamin E (VE), immune cell population analyses were conducted using thymus and spleen from male broilers fed diets with various levels of VE supplementation (0, 17, 46, and 87 mg dl-alpha-tocopherol acetate/kg of feed). At 2 and 7 wk of age, the percentages of B cells, macrophages, and T cell subsets, delineated by the expression of CD4, CD8, and T cell receptor (TCR) isotype, in thymus and spleen were determined by flow cytometry. The percentages of thymic and splenic B cells and macrophages from 2- and 7-wk-old chickens, as well as the percentage of thymic T cells in 2-wk-old chickens, were unaffected by VE treatment. However, 7-wk-old broilers maintained on 87 mg VE/kg feed had a higher percentage of CD4+CD8- thymocytes, a higher CD4+CD8- to CD4-CD8+ thymocyte ratio, and a lower percentage of CD4+CD8+ thymocytes than chickens receiving no dietary VE supplementation. The VE-induced increase in the percentage of CD4+CD8- thymocytes was due to an increase in the TCR2+CD4+CD8- thymocyte subset, whereas the decrease in the percentage of CD4+CD8+ thymocytes involved all TCR defined T cell subsets. In the spleen, the percentage of CD4+CD8- T cells was lower in 2-wk-old chickens and higher in 7-wk-old chickens maintained on 87 mg/kg feed than in chickens receiving no dietary VE supplementation. The decrease in CD4+CD8- splenocytes at 2 wk of age was due to a decline in the percentage of TCR2+CD4+CD8- splenocytes, whereas the increase in CD4+CD8- splenocytes in 7-wk-old chicks was due to an increase in the percentages of all TCR defined CD4+CD8- T cell subsets. These data support an immunomodulatory effect of VE on CD4+CD8- T cells.     

Progressive hearing loss in hard-of-hearing children

Roles of CD8+ and CD4+ cells on lethal graft-versus-host disease (GVHD) were investigated. Injection of spleen cells from C57BL/6 (B6) female mice into (BALB/c x B6)F1 nu/nu female mice caused subacute lethal GVHD (survival: 10-50 days). Injection of anti-Lyt-2.2 (CD8) monoclonal antibody (mAb) on days zero, four and 14 into recipient mice prolonged their survival for at least the 200-day observation period. Injection of anti-L3T4 (CD4) mAb also prolonged survival of the mice for more than 70 days, but they eventually died by 150 days. Pretreatment of the donor B6 spleen cells with anti-Lyt-2.2 (CD8) mAb and complement (C) prevented the development of GVHD, and their pretreatment with anti-L3T4 (CD4) mAb and C markedly prolonged the survival of recipient mice. Injection of a mixture of donor spleen cells pretreated with anti-Lyt-2.2 (CD8) mAb and C and those pretreated with anti-L3T4 (CD4) mAb and C induced subacute lethal GVHD. Injection of anti-L3T4 (CD4) mAb, but not anti-Lyt-2.2 (CD8) mAb on days five, nine and 14 prolonged survival of the recipient mice. These results indicated that the collaboration of CD8+ cells and CD4+ cells was necessary for induction of subacute lethal GVHD. CD4+ cells but not CD8+ cells were involved in mediating subacute GVHD from the onset of the disease. CD8+ cells were, however, capable of inducing late-onset lethal GVHD. Direct phenotyping of T cells in the recipient mice revealed that the CD4+ cells were incapable of repopulating without CD8+ cells, but that CD8+ cells were capable of repopulating without CD4+ cells.     

Histidine-rich glycoprotein binding to T-cell lines and its effect on T-cell substratum adhesion is strongly potentiated by zinc

Fibroblasts have an important structural role in the spleen, as they provide a scaffold of extracellular matrix in which cells of the immune system reside. Aside from their vague recognition as "stromal" or "reticular" components of the spleen, these cells have not been characterized. In this study, normal fibroblast lines from mouse [B6D2(F1)] spleen were established. The fibroblast phenotype of these lines was confirmed by their morphology, expression of vimentin, as well as their lack of epithelial and endothelial cell markers, their failure to display the hematopoietic marker CD45, and their inability to phagocytize. Interestingly, 50-65% of the splenic fibroblasts expressed the Thy-1 antigen, while a subpopulation of Thy-1-negative fibroblasts existed. FACS on the basis of Thy-1, as well as limiting dilution cloning, yielded stable lines and clones of Thy-1+ and Thy-1- splenic fibroblasts. Phenotypic characterization revealed that both subsets synthesized collagen and expressed class I MHC, ICAM-1, VCAM-1, and CD44 constitutively. However, intriguing differences existed between the fibroblast subpopulations. Thy-1+ splenic fibroblasts produced significantly greater levels of IL-6 than did their Thy-1- counterparts. After treatment with IFN-gamma (150 U/ml, 72 hr), Thy-1-, but not Thy-1+, splenic fibroblasts expressed class II MHC and presented antigen to an I-A(b)-restricted T cell line. This suggests that the Thy-1- fibroblasts may present antigen to T lymphocytes in vivo under inflammatory conditions. Thus, splenic fibroblasts are a heterogeneous and dynamic cell type poised in an immunologically relevant location to interact with bone marrow-derived cells under normal and fibrotic conditions.     

IFN-gamma prevents Th2 cell-mediated pathology after neonatal injection of semiallogenic spleen cells in mice

BALB/c mice injected at birth with 10(8) (A/J X BALB/c)F1 hybrid spleen cells develop an autoimmune host-vs-graft (HVG) disease as a result of activation of donor B cells by host CD4+ cells. The antidonor CD4+ cells seem to be Th2-like cells, inasmuch as they are profoundly deficient in IL-2 and IFN-gamma production, but secrete high levels of IL-4 and IL-10. As IFN-gamma is known to inhibit the development of TH2 cells, we attempted to modulate HVG disease by injecting rIFN-gamma. First, we found that 10 micrograms of rIFN-gamma given on days 1 and 3 after birth reduced the serum hyper-IgE of HVG mice by 90% and the serum hyper-IgG1, by 70%. In addition, rIFN-gamma administration significantly decreased the anti-DNA IgG1 titers and prevented the occurrence of anti-glomerular basement membrane and anti-laminin IgG1 Abs as well as the formation of immune deposits in renal glomeruli. These effects were not caused by the abrogation of chimerism, as indicated by the persistence of donor-type B cells in lymph nodes and of Igs bearing donor allotype in serum. MLC experiments indicated that the major effect of early rIFN-gamma administration was to restore the production of IL-2 and IFN-gamma by donor-specific T cells while these cells still secreted significant amounts of IL-4 and IL-10. Unresponsiveness of antidonor cytolytic T cells was not influenced by rIFN-gamma. We conclude that rIFN-gamma prevents the TH2-type response induced by the neonatal injection of semiallogeneic spleen cells and the associated pathology.     

Mucosally induced systemic T cell unresponsiveness to ovalbumin requires CD40 ligand-CD40 interactions

CD40 ligand (CD40L) gene-disrupted (CD40L-/-) mice were employed to examine the role of costimulatory signals via CD40L-CD40 interactions in mucosally induced tolerance. CD40L-/- and control (CD40L+/+) mice of the same C57BL/6 x 129/J background were immunized orally with 25 mg of OVA before systemic challenge with OVA in CFA. While CD40L+/+ mice showed reductions in Ag-specific T cell responses including delayed-type hypersensitivity (DTH) and proliferative responses, CD40L-/- mice underwent normal T cell responses. Further, cytokine analysis of splenic CD4+ T cells showed that both Th1-type (e.g., IFN-gamma and IL-2) and Th2-type (e.g., IL-4, IL-5, IL-6, and IL-10) responses were maintained in CD40L-/- mice orally immunized with OVA, whereas these cytokine responses in CD40L+/+ mice were significantly reduced. In addition, splenic CD4+ T cells from CD40L-/- mice orally immunized with OVA provided B cell help in Ag-specific Ab-forming cells when the cells were cultured with naive B cells in the presence of Ag and CD40L-transfected cell lines. In contrast, an identical culture condition containing splenic CD4+ T cells from orally tolerized CD40L+/+ mice did not exhibit helper activity. Taken together, these findings indicate that CD40L and CD40 interactions are essential for the induction of systemic T cell unresponsiveness to orally administered Ag.