Mouse Mastocytoma P-815 Cells Research Articles

Effect of Sorbic Acid on the Induction of Static Growth Inhibition in Mouse Mastocytoma P-815 Cells

Effect of Sorbic Acid on the Induction of Static Growth Inhibition in Mouse Mastocytoma P-815 Cells

Role of Integrins Involved in Mn2+-Dependent Adhesion to Fibronectin Peptide of Mastocytoma P-815 Cells and Peritoneal Mast Cells

Mn2+-dependent integrin-mediated adhesion to the extracellular matrix is extensively studied, however, its implication in mast cell biology remains unexplored. This study aims to investigate the role of Mn2+ in promoting adhesion in mouse mastocytoma P-815 cells (P-815) and peritoneal mast cells (PMC) to the Arg-Gly-Asp (RGD)-enriched fibronectin peptide (RGD matrix) within the culture medium. Our findings indicate that Mn2+ induces cell adhesion, with optimal results achieved when P-815 were exposed to 2 mM Mn2+ for 30 min at 37ºC, resulting in approximately 40% cell adhesion to the RGD matrix. The Mn2+-dependent P-815 adhesion was inhibited by anti-integrin α4, β1, and β3 subunit function-blocking antibodies, and by the integrin αIIbβ3 antagonist tirofiban, indicating the involvement of integrins α4β1 and αIIbβ3. Similarly, Mn2+-dependent PMC adhesion to the RGD matrix was inhibited by anti-integrin α4, α5, β1, β3, and β7 subunit function-blocking antibodies and tirofiban, demonstrating the involvement of integrins α4β1, α4β7, α5β1, and αIIbβ3. Integrins α4β1 and αIIbβ3 were consistently involved in Mn2+-induced adhesion reactions in both P-815 and PMC, while integrins α4β7 and α5β1 were specifically implicated in the response to PMC only. The addition of the actin inhibitor cytochalasin D, glycosylphosphatidylinositol-anchored protein (GPI-AP) cleaving enzyme phosphatidylinositol-specific phospholipase C, and the PKA inhibitor H-89 significantly reduced Mn2+-dependent P-815 adhesion to the RGD matrix. However, adding the myosin II inhibitor brebbistatin and the RhoA inhibitor Y27632 did not produce the same effect. Furthermore, cellular cholesterol removal with 6-O-α-maltosyl-β cyclodextrin significantly diminished Mn2+-dependent P-815 adhesion, concomitant with a decrease in the expression of integrin α4 and β1 subunits on the cell surface.

Butyrate suppresses murine mast cell proliferation and cytokine production through inhibiting histone deacetylase

Beyond their nutritional impact to colonic epithelial cells, the intestinal microbiota metabolite butyrate has pleotropic effects to host cells and is known for its beneficial effects on intestinal homeostasis and metabolism. However, it remains unclear how it modulates mast cell function. Here, we demonstrate that butyrate profoundly inhibited proliferation of mouse mastocytoma P815 cells through inducing cell cycle arrest and apoptosis, as well as decreasing c-Kit activation. In addition, butyrate increased early- and late-stage apoptotic P815 cells. In murine bone marrow-derived mast cells (BMMC), butyrate-suppressed FcεRI-dependent tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) release without affecting β-Hexosaminidase, but that was associated with decreased mitogen-activated protein kinase extracellular signal-regulated kinase 1/2, p38 and c-Jun N-terminal kinases activation. Butyrate treatment substantially enhanced histone 3 acetylation in both P815 and BMMC and decreased FcεRI-dependent mRNA expression of tnf-α and il-6 in BMMC, mimicking the effect of Trichostatin A, a known histone deacetylase inhibitor. Chromatin immunoprecipitation revealed that butyrate enhanced acetylation of the tnf-α and il-6 promoter regions but blocked RNA polymerase II binding to the promoters of tnf-α and il-6 genes, indicating suppressed transcription initiation. These phenotypes mimicked those of Trichostatin A treatment. In conclusion, butyrate inhibits cell proliferation and increases cell apoptosis in mastocytoma P815 cells and suppresses FcεRI-dependent cytokine production in murine primary BMMC, which are likely mediated by HDAC inhibition.

Effect of 6-O-α-maltosyl-β cyclodextrin and its cholesterol inclusion complex on cellular cholesterol levels and ABCA1 and ABCG1 expression in mouse mastocytoma P-815 cells

We have previously described 6-O-α-maltosyl-β cyclodextrin (Mal-βCD), which forms soluble inclusion complex with cholesterol. Here we further investigated the effect of Mal-βCD and cholesterol/Mal-βCD inclusion complex (CLM) on cellular cholesterol levels in a mouse mast cell line, mastocytoma P-815 cells (P-815 cells). Mal-βCD removes cellular cholesterol forming inclusion complexes, while Mal-βCD-induced lack of cellular cholesterol was replenished by the addition of CLM without cytotoxicity. Reduction and replenishment of cellular cholesterol in Mal-βCD- and/or CLM-treated P-815 cells, respectively, were demonstrated by LC/MS and fluorescence microscopy with filipin III. CLM rather than free Mal-βCD and free cholesterol was efficiently incorporated into P-815 cells and its incorporation was inhibited by incubation at low temperature, or with sodium azide and cytochalasin D. P-815 cells have been confirmed to express ATP-binding cassette (ABC) transporters, ABCA1, ABCG1, and P-glycoprotein (P-gp), by Western blot and mRNA analysis. Cholesterol reduction by Mal-βCD abolishes the mRNA and protein expression of ABCA1 and ABCG1, but not of P-gp. Cholesterol loading by CLM restores the diminished ABCA1 and ABCG1 mRNA expression in Mal-βCD-treated P-815 cells. However, both Mal-βCD and CLM had no effect on P-gp activity measured by the rhodamine 123 efflux assay. These results indicate that alteration of cholesterol levels with Mal-βCD or CLM led to down- or up-regulation of ABCA1 and ABCG1 expression in P-815 cells.

Tumor expression of CD200 inhibits IL‐10 production by tumor‐associated myeloid cells and prevents tumor immune evasion of CTL therapy

CD200 is a cell-surface glycoprotein that functions through interaction with the CD200 receptor on myeloid lineage cells to regulate myeloid cell functions. Expression of CD200 has been implicated in multiple types of human cancer; however, the impact of tumor expression of CD200 on tumor immunity remains poorly understood. To evaluate this issue, we generated CD200-positive mouse plasmacytoma J558 and mastocytoma P815 cells. We found that established CD200-positive tumors were often completely rejected by adoptively transferred CTL without tumor recurrence; in contrast, CD200-negative tumors were initially rejected by adoptively transferred CTL but the majority of tumors recurred. Tumor expression of CD200 significantly inhibited suppressive activity and IL-10 production by tumor-associated myeloid cells (TAMC), and as a result, more CTL accumulated in the tumor and exhibited a greater capacity to produce IFN-gamma in CD200-positive tumors than in CD200-negative tumors. Neutralization of IL-10 significantly inhibited the suppressor activity of TAMC, and IL-10-deficiency allowed TAMC to kill cancer cells and their antigenic variants, which prevented tumor recurrence during CTL therapy. Thus, tumor expression of CD200 prevents tumor recurrence via inhibiting IL-10 production by TAMC.

PGE2-receptor subtype EP4-dependent adherence of mastocytoma P-815 cells to matrix components in subcutaneous tissues overlaying inside surface of air pouch cavity in CDF1 mouse

It remains to be fully clarified how adhesion of mast cells is regulated in vivo. We previously reported that PGE2-receptor EP4 stimulated the adhesion of mouse mastocytoma P-815 cells to plate-bound fibronectin. Our purpose in this study is to evaluate the adhesion using a system, which can mimic the in vivo adhesion. P-815 cells were transplanted in an air pouch produced in the transplantable mice, CDF1. The number of cells that adhere to the subcutaneous tissues overlaying the inside cavity surface was determined. The number of adhered cells was decreased in mice administered with ibuprofen or an EP4 antagonist, ONO AE3-208. A local administration of PGE(2) or a phorbol ester, PMA, increased the number of adhered cells, which was also suppressed in the mice treated with ONO AE3-208. Our results suggest that PGE(2)-mediated adhesion of P-815 cells in the subcutaneous tissues of the air pouch is mediated by the EP4 subtype.

Inhibition by reproterol of cAMP PDE in intact mastocytoma P-815 cells

In vitro studies in rat mastocytes and human monocytes suggested that reproterol (a selective β 2-adrenoceptor agonist with a theophylline moiety) exerts anti-inflammatory actions through inhibition of cyclic AMP (cAMP) PDE activity. Thus, reproterol was tested for its ability to inhibit cAMP PDE in cultured mouse mastocytoma P-815 cells. cAMP PDE activity was measured in intact cells by spectrofluorometry using the fluorescent substrate 2′- O-anthraniloyl cAMP. Reproterol was more potent than theophylline to inhibit cAMP PDE (pIC 50=4.28±0.25 vs. 3.16±0.05). This contrasted with disrupted cells, where the PDE inhibitory potency of reproterol was low (pIC 50=2.85±0.03) and similar to that of theophylline (pIC 50=2.66±0.19). No cAMP PDE inhibition was found with other β 2-agonists tested (fenoterol, salbutamol, salmeterol and formoterol). Finally, the selective PDE inhibitors calmidazolium (100 nM), milrinone (5 μM) and rolipram (50 μM) inhibited cAMP PDE activity by ≈20, 30 and 25% respectively. In conclusion, reproterol potently and non-specifically inhibited intracellular cAMP phosphodiesterases in intact mastocytoma cells. This can explain the previously reported β 2-adrenoceptor-independent anti-inflammatory actions of reproterol in vitro. Further studies are required to define the anti-inflammatory potential of reproterol in asthma.

Histidine Decarboxylase Expression in Human Melanoma

Histamine has been implicated as one of the mediators involved in regulation of proliferation in both normal and neoplastic tissues. Histidine decarboxylase, the only enzyme that catalyzes the formation of histamine from L-histidine, is an essential regulator of histamine levels. In this study, we investigated the gene and protein expression of histidine decarboxylase in melanoma. Reverse transcriptase polymerase chain reaction and in situ hybridization studies of WM-35, WM-983/B, HT-168, and M1 human melanoma cell lines both resulted in positive signals for histidine decarboxylase messenger RNA. A polyclonal chicken antibody was developed against human histidine decarboxylase and protein expression was confirmed by western blot analysis of the cell lysates, revealing a predominant immunoreactive band at approximately 54 kDa corresponding to monomeric histidine decarboxylase. Protein expression of histidine decarboxylase was also shown by flow cytometric analysis and strong punctate cytoplasmic staining of melanoma cell lines. Moreover, both primary and metastatic human melanoma tissues were brightly stained for histidine decarboxylase. When compared with the very weak or no reactions on cultivated human melanocytes both western blot and immunohistochemical studies showed much stronger histidine decarboxylase expression in melanoma cells. These findings suggest that expression of histidine decarboxylase is elevated in human melanoma.

Cellular damage and altered carbohydrate expression in P815 tumor cells induced by direct electric current: an in vitro analysis.

Treatment with direct electric current (DC) can inhibit tumor growth in several systems. To evaluate the cellular reactions generated by this treatment, we stimulated mouse mastocytoma P815 cells with DC and examined their viability and ultrastructural characteristics, as well as the effect of DC on surface carbohydrate expression. DC treatment affected cell viability and caused marked alterations in vital structures of P815 cells. Alterations varied depending on the duration of stimulation and polarity of electrode. Anodic and cathodic treatments caused decrease in cell viability, although the latter was more effective in generating cell lysis. DC stimulation also induced changes such as membrane damage, alterations in cell shape and chromatin organization, mitochondrial swelling and condensation, cytoplasmic swelling, and matrix rarefaction. Stimulation of P815 cells without contact with electrodes produced no alterations, suggesting that this contact might be essential for the occurrence of the cellular modifications. DC treatment also altered the membrane distribution of anionic sites of P815 cells, as well as the surface carbohydrate exposition, involving a diminished binding of Concanavalin A to the cell surface after cathodic stimulation, and an increased binding of sialic acid- and fucose-specific lectins after anodic treatment. In this work we describe important cellular targets for the action of DC, which may contribute to the understanding of the mechanisms by which DC supresses several kinds of tumors.

Inhibition of kit expression in P815 mouse mastocytoma cells by a hammerhead ribozyme.

Using P815 cells, we designed and tested an antisense hammerhead ribozyme for its ability to mediate cleavage of mouse c-kit mRNA at the transmembrane-encoding region. The ribozyme demonstrated in vitro cis cleavage of extraneous 5' sequence in the ribozyme construct as well as trans cleavage of a target c-kit mRNA sequence. The in vitro cleavage was effective at 37 degrees C. A subpopulation of P815 cells transfected with the pCDNA1 plasmid-containing the anti-c-kit ribozyme demonstrated inhibited Kit expression as indicated by flow cytometry, but the inhibition could not be maintained over time. The anti-c-kit ribozyme was put under the control of a tetracycline-inducible two-plasmid system of expression. Incomplete inhibition of Kit expression was observed when transcription of the ribozyme was allowed by the absence of tetracycline, but not when tetracycline was present. Finally, complete inhibition of Kit expression was observed when pCDNA1-ribozyme-transfected cells were analyzed immediately after enrichment by cotransfection with a plasmid expressing green fluorescent protein, followed by cell sorting. The results suggest that anti-c-kit ribozymes might be useful for inhibiting Kit expression in hyperplastic mast cells, if delivery of ribozymes can be optimized.

INTERLEUKIN-3 OR IMMUNOGLOBULIN E PROMOTES EXPRESSION OF PROTEIN KINASE C δ GENE IN MURINE MAST CELLS

We reported previously that protein kinase C δ (PKCδ) is the main isoenzyme in various types of murine mast cells. In the present study we investigated the regulation of expression of PKCδ gene in murine mast cells in vitro and in vivo. The mRNA expressions of PKCδ were promoted in response to interleukin-3 (IL-3) or immunoglobulin E (IgE) in mouse mastocytoma P-815 cells. In addition we have evaluated the mast cells which express PKCδ mRNA in IgE-dependent passive cutaneous anaphylaxis reaction, using in situ hybridization with the antisense riboprobe in skin. These results indicate that mast cell activation can induce a marked promotion in steady state levels of PKCδ mRNA.1999 Academic Press@p$hr

Response of fish immune cells to in vitro organotin exposures

The presence of contaminants in aquatic environments may compromise the health and survival of fish. Many of these compounds are known immunotoxins in mammals; however, relatively little information is available on the immunotoxic responses of fish to these pollutants. Organotins are examples of widespread industrial and agricultural compounds which persist in aquatic environments. In this study, we investigate the effects of tributyltin (TBT) and its dealkylated metabolite dibutyltin (DBT) on fish immune responses. Immune cells were isolated from the spleen and head kidney of juvenile rainbow trout ( Oncorhynchus mykiss) and exposed to 0, 2.5, 50 and 500 ppb of either TBT or DBT. Mitogenesis was quantified by tritiated thymidine incorporation into cells cultured with the mitogens concanavalin A (Con A) or lipopolysaccharide (LPS). No changes in immune function occurred at the lowest organotin dose of 2.5 ppb. Con A-stimulated mitogenesis was significantly suppressed by 85% in spleen cells on exposure to 50 ppb DBT. LPS-stimulated mitogenesis was significantly suppressed by 96% in spleen cells and by 58% in head kidney cells with 50 ppb DBT. The highest concentration of 500 ppb of TBT and DBT inhibited both Con A- and LPS-stimulated mitogenesis by more than 95% in both head kidney and spleen cells. Flow cytometric analysis revealed dose-dependent changes in the cell population profile which correlated with the inhibition of mitogen-stimulated lymphoproliferation. In contrast, natural cytotoxic cell activity was not inhibited by in vitro exposure to either compound, as determined by the lysis of chromium-51-labelled K562 human erythroleukaemia cells and P815 mouse mastocytoma cells. These results show that organotins have both functional and tissue-specific effects on the fish immune system, i.e. spleen ⪢ head kidney tissue and, in general. LPS-responsive ⪢ Con A-responsive leukocytes. In aquatic systems, TBT is considered to be the most toxic organotin compound, and this toxicity decreases with progressive dealkylation to diand mono-organotins. However, our results indicate that DBT is a more potent immunotoxin than TBT, and suggest a need for the reassessment of the potential toxicity of DBT to aquatic organisms.

Novel histamine measurement by HPLC analysis used to assay histidine decarboxylase inhibitory activity of shoyuflavones from soy sauce.

An easy and highly sensitive method for measuring histamine by HPLC analysis coupled with precolumn derivatization was established. The amino group of histamine was completely colorimetrically labelled with 4-N,N-dimethylamino-azobenzene-4'-isothiocyanate (DABITC) in the presence of sodium bicarbonate at 90 degrees C for 5 min. The derivative was sensitively and easily analyzed by HPLC on a Cosmosil 5SL column using CHCl3/N,N-dimethylformamide/H2O (210:90:4) containing 0.4% acetic acid. Using the established method, histidine decarboxylase (HDC) inhibitory activities of three tartaric acid isoflavone derivatives, named shoyuflavones, isolated from soy sauce were examined in vitro by measuring the histamine produced by HDC. They showed intense inhibition of the activities of HDC from both mouse mastocytoma P-815 cells and Clostridium perfringens.

Solanum Lyratum Inhibits Anaphylactic Reaction and Suppresses the Expression of L- Histidine Decarboxylase mRNA

We investigated the effect of aqueous extract of Solanum lyratum Thunb. (Solanaceae) (SLAE) on anaphylactic reaction. The mast cell is widely thought to contribute to the acute changes associated with anaphylaxis. SLAE inhibited skin mast cells-mediated anaphylactic reaction activated by anti-dinitrophenyl (DNP) IgE. SLAE dose-dependently inhibited histamine release in mouse peritoneal mast cells activated by anti-DNP IgE or substance P. Substance P increased steady state levels of L-histidine decarboxylase (HDC) mRNA in mouse mastocytoma P-815 cells. Northern-blot analysis demonstrated that significantly reduced level of the mRNA of HDC was expressed in mast cells treated with SLAE, compared to that without SLAE. We conclude that SLAE directly affect IgE-mediated anaphylactic reaction and substance P-induced HDC mRNA over-expression.

Isolation and characterization of two distinct lectins with antiproliferative activity from the cultured mycelium of the edible mushroom Tricholoma mongolicum.

Two lectins, TML-1 and TML-2, were isolated from Tricholoma mongolicum by ion-exchange chromatography and gel filtration. They are dimers with molecular weight near 37000. The hemagglutinating activities of TML-1 and TML-2 are sensitive to lactose inhibition and are stable between 10 and 80 degrees C. They exhibit antiproliferative activities against mouse monocyte-macrophage PU5-1.8 cells and mouse mastocytoma P815 cells in vitro. The two lectins differ in the content of proline and tyrosine residues. Both are non-glycoproteins and have hydroxyproline residues.

Production of nitric oxide by rat type II pneumocytes: increased expression of inducible nitric oxide synthase following inhalation of a pulmonary irritant.

Nitric oxide is a highly reactive molecule that has been implicated in host defense and tissue injury. In the present studies, we determined whether rat type II alveolar epithelial cells have the capacity to produce this mediator. We found that type II cells synthesize significant quantities of nitric oxide after treatment with the inflammatory cytokines, interferon-gamma (IFN-gamma) and/or interleukin-1 beta (IL-1 beta), or with the combination of IFN-gamma and tumor necrosis factor-alpha. In contrast to rat alveolar macrophages, type II cells were unresponsive to lipopolysaccharide. Production of nitric oxide by type II cells in response to IFN-gamma was dose dependent, reaching a maximum at 100 U/ml, and blocked by NG-monomethyl-L-arginine (L-NMA), a nitric oxide synthase inhibitor. Northern blot analysis demonstrated that nitric oxide production by type II cells was due to expression of mRNA for an inducible form of nitric oxide synthase (iNOS). Following brief exposure of rats to irritant-inducing doses of ozone (2 ppm, 3 h), type II cells were found to produce significantly more nitric oxide than were cells from control animals. This was due to increased expression of iNOS mRNA. Cells from ozone-treated rats were also sensitized to produce more nitric oxide in response to IFN-gamma and IL-1 beta. This was associated with a marked increase in expression of iNOS mRNA and enzyme protein in the cells. We also found that ozone inhalation caused enhanced production of hydrogen peroxide, as well as spontaneous and IFN-gamma-induced cytostasis of type II cells toward P815 mouse mastocytoma cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell-mediated cytotoxicity is protected in moderately iron-deficient C57BL/6 mice

Severe iron-deficiency anemia impairs cell-mediated cytotoxicity; however, the effect of milder degrees of iron deficiency on cell-mediated cytotoxicity has not been characterized. T-cytotoxicity against P815 mouse mastocytoma cells (Experiment 1) and natural killer (NK) cytotoxicity against Yac-1 mouse lymphoma and S194 mouse myeloma cells (Experiment 2) were measured in spleen lymphocytes prepared from control and mildly anemic iron-deficient C57BL/6 mice. Weanling mice (n=10) were fed diets ad libitum with 48 mg iron/kg (control), 5 or 7 mg iron/kg (iron-deficient), or were pair-fed 48 mg iron/kg diet at the level consumed by iron-deficient mice (pair-fed) for 50–54 d. In Experiment 1, mice were sensitized by an intraperitoneal injection of 1 X 10 7 P815 cells at 10 and 5 days prior to sacrifice. In Experiment 2, mice were given an injection of polyinosinic:polycytidylic acid (poly I:C; 10 μg/g body weight) 24 hr prior to sacrifice to augment NK activity. Blood hemoglobin was significantly lower in iron-deficient mice (Experiment 1, 102±18 g/l; Experiment 2, 106±6 g/l) fed 5 or 7 mg iron/kg diet compared with control mice (Experiment 1, 146±10 g/l; Experiment 2, 165±8 g/l) and pair-fed mice (Experiment 1, 151±14 g/l; Experiment 2, 136±5 g/l) fed 48 mg iron/kg diet. Pair-fed mice were fed 16.8% (Experiment 1) or 16.4% (Experiment 2) less food than consumed ad libitum by control mice and had significantly lower body weight compared with control mice. In both experiments, cytotoxicity of spleen lymphocytes (effector cells) against target cells (P815, Yac-1, S194) was measured by a chromium-51 release assay at effector:target cell ratios (E:T) of 50, 25, and 10:1. Spleen lymphocytes from control and iron-deficient animals displayed similar levels of both T-cytotoxicity and NK cytotoxicity, while lymphocytes from pair-fed animals displayed greater levels of both types of cytotoxicity. Results of this study demonstrate that cell-mediated cytotoxicity is preserved in moderate iron deficiency accompanied by mild anemia, while it is enhanced by a modest food restriction in the absence of micronutrient deficiency.

Effects of subunit mutation on the localization to coated pits and internalization of cross-linked IgE-receptor complexes.

IgE receptors of mast cells, Fc epsilon RI, localize to coated pits and internalize after cross-linking. We investigated whether any one of the receptor's four distinctive cytoplasmic domains regulates these phenomena. COS cells, which lack Fc epsilon RI entirely, and P815 mouse mastocytoma cells that lack the alpha and beta subunits of the tetrameric Fc epsilon RI (alpha beta gamma 2), were transfected with wild-type, incomplete, or variant Fc epsilon RI. IgE-receptor complexes were observed by electron microscopy. Before cross-linking with anti-IgE gold particles, receptors were not preferentially localized to coated pits, which occupy approximately 1% of the cell surface. After cross-linking, up to 10 to 20% of the wild-type and most other receptor variants were in coated pits in transfected P815 cells at any one time. beta-less variants localized normally but, surprisingly, receptors containing a variant beta subunit showed reduced localization. "Receptors" consisting simply of the lipid-anchored ectodomains of the human alpha subunit failed to localize to coated pits. In general, cross-linked receptors that localized to coated pits were progressively internalized, whereas receptors that failed to accumulate in coated pits were not. We conclude that no single cytoplasmic domain of the Fc epsilon RI uniquely controls its ligand-induced localization to coated pits and internalization.

12-O-Tetradecanoylphorbol-13-acetate stimulates the transcription of the histidine decarboxylase gene

12-O-Tetradecanoylphorbol-13-acetate (TPA), a potent tumor-promoting phorbol ester, stimulates histidine decarboxylase (HDC) activity in mouse mastocytoma P-815 cells. Activation of a transcriptional factor is believed to be involved in the effect of TPA.In vitro experiments indicated that TPA-induced HDC activation is caused by transcriptional activation of the HDC gene through the TPA-responsive element, TRE.

Simultaneous ligation of CD5 and CD28 on resting T lymphocytes induces T cell activation in the absence of T cell receptor/CD3 occupancy.

The T cell surface molecules CD5 and CD28 have been shown to be receptors for accessory signals in T cell activation. We here demonstrate that in the absence of any other activating stimulus, simultaneous ligation of CD5 and CD28 by mAb induces polyclonal T cell activation. Immobilization of the anti-CD28 and anti-CD5 mAb was an essential requirement for T cell stimulation. This was done either through coating of the culture plates with goat anti-mouse Ig, or by coculture with mitomycin C-treated Fc gamma R-bearing P815 mouse mastocytoma cells. Most importantly, T cells could also be stimulated with B7, the natural ligand of CD28, and anti-CD5 presented on irradiated 3T6 mouse fibroblasts co-transfected with human Fc gamma RII and with B7. Neither immobilized mAb 9.3 (anti-CD28) nor any of four different anti-CD5 mAb were mitogenic as a sole stimulus. Immobilized mAb identifying CD4, CD7, or LFA-1 were not co-mitogenic with either mAb 9.3 or one of the anti-CD5 mAb. The T cell proliferation induced by cross-linking of CD5 and CD28 is IL-2-dependent, as was demonstrated by the cell-surface expression of the p55 chain of the IL-2R, the production of IL-2, and inhibition of the proliferative response by the anti-IL-2R mAb anti-Tac. CD5/CD28 ligation induced production of TNF-alpha, but not of IL-4, and did not induce modulation of the TCR/CD3 complex. Expression of IL-2R (p55) and of CD69 preferentially occurred on CD29-low naive cells, and indicated that about 50% of the cultured cells were activated. Cell proliferation was not increased by adding monocytes to the cultures and it was inhibited by PKC inhibitors (H7 and staurosporine) and by cyclosporine A. In conclusion, our data provide evidence for a pathway of Ag-independent T cell activation via CD5 and CD28, which preferentially stimulates native T cells.