T cell regulation of the immune response to infection in periodontal diseases

Although T cells have been implicated in the pathogenesis and are considered to be central both in progression and control of the chronic inflammatory periodontal diseases, the precise contribution of T cells to the regulation of tissue destruction has not been fully elucidated. Current dogma suggests that immunity to infection is controlled by distinct T helper 1 (Th1) and T helper 2 (Th2) subsets of T cells classified on the basis of their cytokine profile. Further, a subset of T cells with immunosuppressive function and cytokine profile distinct from Th1 or Th2 has been described and designated as regulatory T cells. Although these regulatory T cells have been considered to maintain self-tolerance resulting in the suppression of auto-immune responses, recent data suggest that these cells may also play a role in preventing infection-induced immunopathology. In this review, the role of functional and regulatory T cells in chronic inflammatory periodontal diseases will be summarized. This should not only provide an insight into the relationship between the immune response to periodontopathic bacteria and disease but should also highlight areas of development for potentially new therapeutic modalities.     

Antibody-mediated enhancement of infection by dengue virus of the P815 murine mastocytoma cell line

Dengue type 2 virus (DEN 2) could replicate only to a limited extent in a murine mastocytoma cell line, P815. The viral multiplication was enhanced 10- to 100-fold by mouse anti-DEN 2 antiserum or anti-DEN 2 type-specific monoclonal antibody diluted beyond their neutralizing titers. Cells incubated with virus-antibody mixtures changed morphologically, developing a mature mast cell-like appearance, 4-5 days after infection. The indirect fluorescent antibody technique showed that the enhancement of infection was caused by an increase in the number of DEN 2-infected cells. This is the first report that cells of mast cell lineage support dengue virus multiplication, and that virus production is enhanced in the presence of anti-dengue antibodies.     

Suppression of cell-mediated immunity to challenge with P 815 mastocytoma in concanavalin A-treated mice

C57Bl/6 (B6) mice allogeneic to the P 815 mastocytoma tumor cell line when treated with concanavalin A prior to and at frequent intervals following challenge intraperitoneally with 10⁷ tumor cells showed a significant suppression of their cell-mediated immune response at 9–10 days when compared with untreated animals. Suppression of the immune response of mice syngeneic (DBA/2) or hybrid (BDF₁) to the tumor was also evidenced by increased mortality rates in concanavalin A-treated animals. The suppression of cell-mediated cytotoxicity observed in B6 mice treated with concanavalin A could be reversed by pretreatment with 20 mg silica injected intraperitoneally 7 days prior to challenge. These results suggest that macrophages play a significant role in the concanavalin A-induced immune suppression observed in this in vivo tumor-host system.     

Thapsigargin induces mitochondrial dysfunction and apoptosis in the mastocytoma P815 cell line and in mouse thymocytes

Thapsigargin is a plant-derived inhibitor of the endoplasmic reticulum Ca(2+)-ATPase.Treatment with thapsigargin leads to a rapid, large and prolonged increase in the intracellular calcium ion concentration ([Ca(2+)](i)). Previously thapsigargin has been shown to inhibit proliferation and induce apoptosis. Here we report the results of thapsigargin treatment in thymocytes harvested from 10-day-old mice and in the P815 mastocytoma cell line. In thapsigargin-treated cells we observed enlarged mitochondria with disrupted cristae structure. These mitochondria closely resembled those observed after the induction of phase transition. To determine if the mitochondria were functioning normally the cells were stained with rhodamine 123 (R123) and analysed with flow cytometry. After thapsigargin treatment the R123 staining decreased, indicative of a loss of mitochondrial membrane potential. Furthermore intracellular ATP concentrations were also found to be reduced in cells treated with thapsigargin. Taken together these results indicate an increase in the [Ca(2+)](i) caused by thapsigargin treatment results in dysfunctional mitochondria and reduced ATP. We propose that this decrease in the concentration of ATP provokes the onset of thapsigargin-induced apoptosis. To investigate the effect of thapsigargin treatment on the cell cycle, rapidly cycling P815 cells were sorted into populations enriched for either G(0)/G(1) or S/G(2)/M phases, and these populations were then treated with thapsigargin. Thapsigargin treatment induced a cell cycle block before S phase. We propose that the block in the cell cycle induced by thapsigargin was a result of the decreased intracellular ATP concentration interfering with the energy requiring processes of DNA replication. The block could also be related to the high intracellular calcium ion concentration that would interfere with the subtle calcium transients involved in the cell's preparations for replication and mitosis. Apoptosis occurred to an equal extent in both populations of cells.     

Characterization of cytosolic pertussis toxin-sensitive GTP-binding protein in mastocytoma P-815 cells

We have characterized a soluble pertussis toxin (PT)-sensitive GTP-binding protein (G-protein) present in mouse mastocytoma P-815 cells. 65% of total ADP-ribosylation of PT substrate having a molecular mass of 40 kDa on SDS-polyacrylamide gel electrophoresis in cell homogenate was detected in the supernatant after centrifugation at 100,000 x g for 90 min. [32P]ADP-ribosylation of cytosolic PT substrate was significantly enhanced on the addition of exogenous beta gamma complex. The molecular mass of the cytosolic PT substrate was estimated to be about 80 kDa on an Ultrogel AcA 44 column, but the beta gamma complex was not detected in the cytosol by using the anti-beta gamma complex antibody. Furthermore, the cytosolic PT substrate was found to have some unique properties: [35S]GTP gamma S binding was not inhibited by GDP and [32P]ADP-ribosylation was not affected by GTP gamma S treatment. Only after the cytosolic PT substrate had been mixed with exogenous beta gamma complex, did it copurify with exogenous beta gamma complex by several column chromatographies including an Octyl-Sepharose CL-4B column. The PT substrate was identified as Gi2 alpha by Western blot analysis and peptide mapping with S. aureus V8 protease. These results suggest that Gi2 alpha without beta gamma complex exists with an apparent molecular mass of about 80 kDa in the cytosolic fraction of P-815 cells.     

Inhibitory effects of tetrahydropapaverine on serotonin biosynthesis in murine mastocytoma P815 cells

The inhibitory effects of tetrahydropapaverine on serotonin biosynthesis in serotonin-producing murine mastocytoma P815 cells were investigated. Tetrahydropapaverine at concentration ranges of 5-20 microM decreased serotonin content in a concentration-dependent manner in P815 cells and showed 42.1% inhibition of serotonin content at 5.0 microM at 24 hr. The value of 50% inhibitory concentration, IC50, of tetrahydropapaverine was 6.2 microM. Under these conditions, tryptophan hydroxylase (EC 1.14.16.4, TPH) was inhibited for 24-36 hr after treatment with tetrahydropapaverine in P815 cells (49.1% inhibition at 7.5 microM). However, aromatic L-amino acid decarboxylase activity was not affected by tetrahydropapaverine. In addition, tetrahydropapaverine inhibited the activity of TPH, prepared from the P815 cells (P815-TPH), with the IC50 value of 5.7 microM. Tetrahydropapaverine un-competitively inhibited P815-TPH with the substrate L-tryptophan, and non-competitively with the cofactor DL-6-methyl-5,6,7,8-tetrahydropteridin. The Ki value of tetrahydropapaverine with L-tryptophan was 10.1 microM. These data indicate that tetrahydropapaverine leads to a decrease in serotonin content by the inhibition of TPH activity in P815 cells.     

Role of microvilli in surface changes of synchronized P815Y mastocytoma cells

The surface morphology of synchronized P815Y mastocytoma cells has been examined by scanning electron microscopy. Early G1 cells are comparatively smooth or light villated, whereas at later stages the surface becomes progressively more villated. In G1 cell most microvilli have a uniform diameter, whereas in S and G2 cells, many microvilli show branching and often originate from much larger surface protuberances. Small "blebs" are seen on the surface of many cells but these structures do not appear to be a characteristic feature of cells at any one stage of the cycle. The presence of microvilli increases the total surface of the cell to such an extent that the ratio of volume to surface area remains constant throughout the cell cycle. The mechanism of cytokinesis is thus a physical one, involving the unfolding of previously accumulated microvilli.     

Inhibition of thymidylate synthase by hydroxyurea in rapidly proliferating P815 mastocytoma cells

The incorporation of [¹⁴C]deoxycytidine, [³H]deoxyuridine, and [³H]thymidine, respectively into pyrimidine bases of DNA has been measured in rapidly proliferating P815 mouse mastocytoma cells in the presence of hydroxyurea. The incorporation of [¹⁴C]deoxycytidine-derived radioactivity into DNA cytosines is increased when compared to the incorporation into DNA thymines. The [³H]deoxyuridine-derived radioactivity is incorporated solely into DNA thymines and this incorporation is inhibited by hydroxyurea in a dose-dependent manner. This suggests an inhibitory effect of hydroxyurea on the thymidylate synthase which was proved in experiments in which the conversion of deoxyuridine monophosphate into deoxythymidine monophosphate catalysed by a crude enzyme preparation from P815 cells was inhibited in the presence of hydroxyurea. Enzymatic DNA methylation as measured by the conversion of incorporated [¹⁴C]deoxycytidine into 5-methylcytosines was not affected by hydroxyurea.     

The regulation of immune responses to multiple non-H-2 antigens on tumor cells: I. Differential responses of BALB/c F1 hybrids to the P815 mastocytoma in vivo

Factors influencing host resistance to the growth of a tumor bearing many mismatched minor histocompatibility antigens (MiHA) were studied. BALB/c (H-2^d) and several of its F₁ hybrids were injected intraperitoneally with DBA/2 (H-2^d) P815 tumor cells. Compared to BALB/c, which was moderately susceptible, F₁ hybrids of BALB/c with CBA, AKR, C3H.OH, and BIO H-2-congenic strains were highly susceptible, whereas hybrids of BALB/ c with A, A.SW, and BALB.B strains were quite resistant. Susceptibility was observed only with the intraperitoneally injected tumor, since both BALB/c and (CBA x BALB/c)F₁ were resistant to the same tumor injected subcutaneously, and survival times of DBA/2 skin grafts did not differ between susceptible and resistant strains. Susceptibility was in part a function of the number of MiHA incompatibilities between tumor and host although the specific loci involved could not be identified. For example, susceptible (CBA x BALB/c)F₁ hybrids probably shared certain MiHA with DBA/2 which BALB/c lacked, and which therefore subtracted from the net antigenic strength of the tumor in the hybrid, compared to its strength in BALB/ c. This interpretation was supported by in vitro studies which confirmed that the susceptible hybrids shared more MiHA with DBA/2, than did the resistant hybrids. Resistance was at least partially regulated by the host H-2 genotype, as shown by the observation that (BALB/ c x BALB.B)F₁ (H-2^d/b) mice were significantly more resistant than BALB/c. Segregation studies of the resistant (BALB/c x A)F₁ hybrids, indicated that in addition to H-2, a nonH-2 gene in the A background was operating to confer resistance. Thus the factors influencing susceptibility to the MiHA-incompatible tumor were: (i) site of injection; (ii) the combined strength of the disparate MiHA; (iii) the host H-2 genotype; and (iv) at least one host nonH-2 gene conferring increased responsiveness.     

T cells produce TRF in response to Con A and factors in T cell hybridoma supernatants

In recent experiments, a soluble factor (TRF) that mediates the differentiation of anti-immunoglobulin (Ig)-activated B cells to Ig-secreting cells has been identified. TRF works in concert with a growth factor, probably IL 2, in the induction of activated B cells. In previous studies, TRF was identified in culture supernatants of activated T cells and accessory cells, and thus the cellular source (T cell or accessory cell) of the factor was not determined. In the present studies, we succeeded in inducing the production of TRF by T cell populations from which accessory cells had been vigorously depleted. Lymph node cells were depleted of accessory cells by nylon wool adherence and anti-Ia and complement treatment; these cells were activated with Con A and a T cell hybridoma supernatant that contains IL 2. Supernatants from these activated T cell cultures supported the differentiation of anti-Ig-activated B cells to Ig secreting cells. These results show that T cells produce the differentiation factor, and further that they do so in response to ligand (Con A) plus a T cell-derived factor.     

Suppression of the immune response to ovalbumin in vivo using anti-idiotypic antibodies

The effect of anti-idiotype antibodies (a-IdpI) on primary immune response to ovalbumin (OVA) was studied. A-IdpI against OVA antibodies were obtained with pI 6.2-6.7 from BALB/c mice. About 30-40% of IgG plaque cell formation (PCF) was inhibited if a-IdpI antiserum was added in situ, which served as a test for the evaluation of idiotype-positive (Id+) PCF. Up to 70% of common PCF and 70-80% of Id+ PCF were suppressed in mice that were injected with a-IdpI antibodies prior to immunization. This suppression was antigen- and allo-specific and depended upon the time of a-IdpI injection. If a-IdpI antibodies were disaggregated (DA) the Id+ suppression increased. A-IdpI antibodies also decreased IgE response to OVA, the suppression being most pronounced with the use of DA samples.     

Humoral mechanisms of the membrano-toxic effects of mastocytoma P815 and leukemia EL4 cells

The membranotoxicity activity of P815 and EL4 tumor cells and their humoral factors containing in cultural supernatants and ascitic fluids was estimated. Tumor cells and their humoral factors exert dose-dependent membranotoxicity effect on murine splenocytes and lymphoblasts. Normal murine splenocytes and renal cells have no membranotoxicity properties. Thus tumor cells membranotoxicity effect is carried out by humoral mechanism.     

Humoral mechanisms of in vitro inhibition of mouse lymphocyte immunoreactivity by mastocytoma P815 cells

The inhibitory capacity of mastocytoma cell line P815 and its cultural supernatant (CS) was studied in the reaction of blast transformation (RBT) and mixed lymphocyte culture (MLC). An addition of both P815 cells and CS resulted in dose-dependent inhibition of lymphocyte proliferation in RBT and MLC. The treatment of DBA/2 spleen cells with CS for 2 h at 37 degrees C resulted in a significant decrease in proliferative activity and induction of supressor cells.     

Immune response to the H-X antigen on P815-X2

In a preceding report, the detection of an H-2-linked immune response to the H-X ^d antigen on the P815-X2 mastocytoma was demonstrated by the significantly increased survival of (C57BL/6 × DBA/2)F₁ (B6D2F1) male hybrids (H-X ^b ) compared with female siblings (H-X ^{b/H-X d} ) after injection with the histocompatible tumor (H-X ^d ). This interpretation was supported by the absence of this sex effect in reciprocal D2B6F1 hybrids (H-X ^d and H-X ^{d/H-X b} ). Additional findings presented in this paper support the conclusion that this sex effect is due to a true immunological response to H-X ^d : (a) Reciprocal (DBA/2 × C57BL/6 H-2 mutant)F₁ hybrids, as well as D2B6F1, failed to exhibit the sex effect: (b) the demonstration of the sex effect in (BALB/c × DBA/2)F₁ and (BALB/c-H-2 ^dm2 × DBA/2)F₁ hybrids and in (C57BL/10 × DBA/2)F₁ hybrids was consistent with the known H-X incompatibilities between the strains BALB/c and DBA/2 and C57BL/10 and DBA/2, respectively, previously demonstrated by skin grafting; and (c) the sex effect was not abrogated by castration of male B6D2F1 hybrids. Variability in the presence or absence of the sex effect was observed in various [recombinant inbred (RI) × DBA/2]F₁ hybrids and may be attributed to the influence of a regulatory non-H-2 gene which is closely linked to the gene coding for mouse kidney-androgen-regulated protein (KAP) but androgen-independent, or to variability in inheritance of the H-X ^b allele among the RI lines. It is proposed that the P815-X2 model may be utilized to type RI lines derived from a cross between C57BL/6 and DBA/2 for their H-X genotypes.Abbreviations B C57BL/6 origin allele - B6 C57BL/6 - B10 C57BL/10 - B6D2F1 (C57BL/6 × DBA/2)F₁ - B6 ^m D2F1 (C57BL/6 H-2 mutant × DBA/2)F₁ - bm10 B6.C-H-2 ^bm10 - C BALB/c - D DBA/2 origin allele - D2 DBA/2 - dm2 BALB/c-H-2 ^dm2 - H-X X chromosome-determined histocompatibility antigen of the mouse - Ir gene, immune response gene - KAP kidney androgenregulated protein - MST median survival time - RI recombinant inbred - SDP strain distribution pattern     

Distribution pattern and enzymic hypermethylation of inverted repetitive DNA sequences in P815 mastocytoma cells.

A specific class of DNA sequences, the inverted repetitive sequences, forms a double-stranded structure within a single linear polynucleotide chain in denatured DNA. The reassociation process is unimolecular and occurs very fast. Quantitative analyses have shown that in mouse P815 cells these sequences comprise about 4% of the nuclear DNA and are interspersed within sequences of other degrees of repetitiveness. After labeling the cells with L-[Me-3H]methionine and [14C]deoxycytidine, relative rates of enzymic DNA methylation were computed on the basis of radioactivities found in pyrimidine residues of the nuclear DNA. The results indicate that in P815 cells, DNA of inverted repetitive sequences is methylated to a level about 50% higher than the normal repetitive DNA sequences and to about 300% higher than the unique and intermediary intermediatry sequences. The biological function of the inverted repetitive sequences, as well as of the role of enzymic methylation of DNA remains unknown.     

Immunization of DBA/2 mice with a T cell hybridoma-derived TsF increases immune resistance to the syngeneic tumors P815 and L1210

The ability of a tumor-specific T suppressor factor (TsF) isolated from a T cell hybridoma, A10, to act as an immunogen in DBA/2 mice was investigated. The TsF was affinity purified from ascites over an immunoadsorbent column containing a monoclonal antibody (B16G) that has specificity for the TsF molecule, or over columns containing membrane extracts of the P815 mastocytoma (the tumor for which A10 is specific). The specificity control was BW5147 (the fusion partner for A10) membrane extracts treated in the same way as A10. DBA/2 mice were immunized with the affinity-purified material or PBS and were subsequently challenged with either the P815 tumor or the L1210 DBA/2 thymoma. When mice were immunized with material affinity purified over B16G, eluted material from both A10 ascites and BW5147 membrane extracts enhanced resistance to both P815 and L1210 challenge, indicating that B16G was binding immunogenic material derived from both preparations, which exerted a tumor-protective effect. However, when a P815 affinity column was used, protective material was eluted only from A10 ascites, and this bestowed resistance to both P815 and L1210. When irradiated whole cells were used as immunogens, only A10 cells stimulated anti-tumor immunity, and this appeared to be directed specifically to the P815 tumor. The implications of these findings in terms of the potential for immune modulation with anti-suppressor therapy, and the specificity of the B16G monoclonal, are discussed. The demonstration of B16G binding material (TsF) in the membranes (but not the ascites) of the BW5147 line is also of significance to investigators using BW5147 fused suppressor hybridomas.