Helper signals in the plaque-forming cell response to protein-bound haptens.

We have demonstrated the ability of a series of murine T cell hybridomas to deliver an antigen-specific, B cell I-region-restricted helper signal in the generation of specific PFC responses to protein-bound haptens. With some hybridomas the elicitation of optimal PFC responses required the addition of nonspecific factors provided by culture supernatants of concanavalin A-stimulated (Con A SN) spleen cells. Using hapten-primed B cells depleted of both T cells and macrophages (Mphi) we have now demonstrated a requirement for three nonspecific factor preparations to substitute for spleen Con A SN in the elicitation of optimal PFC responses. The first preparation was the interleukin 1 containing culture supernatant of the Mphi tumor cell line P388D1, the second the interleukin 2 (IL-2) and B cell growth factor containing Con A SN of the T cell hybridoma FS6-14.13, and the third, the gamma interferon containing Con A SN of the T cell hybridoma FS7-20.6.18. The P388D1 and FS6-14.13 factor preparations were most effective when added at the initiation of culture, while the FS7-20.6.18 factor preparation was most effective when added at 24 h of culture. The activity of FS6-14.13 Con A SN was depleted by incubation with the IL-2-dependent T cell line HT-2. The activity of FS7-20.6.18 Con A SN was abrogated by incubation at pH 2. The results suggest that the generation of PFC responses to protein-bound haptens require at least three nonspecific factors in addition to an antigen/Ia specific helper signal.

Antigen-specific, H-2-restricted helper T cell hybridomas.

We have examined the carrier-specific helper activity of a number of antigen-specific, I region-restricted T cell hybridomas prepared in our laboratory. The hybridomas were assayed for helper activity in the presence or absence of exogenously added nonspecific factors found in the concanavalin A-activated supernatants of normal mouse spleen cells. Of six hybridomas tested, all six could stimulate the IgM anti-hapten response of hapten-primed B cells in the presence of the appropriate hapten-carrier conjugates. At low or moderate carrier doses, the response was dependent upon hapten-carrier linkage and the ability of the hybridoma cells to interact with carrier in association with H-2 products of the responding B cells themselves. Plaque-forming cell responses stimulated by some of the hybridomas were absolutely dependent upon the addition of nonspecific factors, suggesting that anti hapten-protein responses require both an antigen specific I region restricted signal from the T cell hybridomas and nonspecific helper factors, made either by the T cell hybridomas or added exogenously. Under two sets of circumstances, B cells were stimulated in the absence of a simultaneous signal delivered through their immunoglobulin receptor. This occurred either when hapten-primed B cells were stimulated with an ovalbumin/I-Ak-specific hybridoma in the presence of very high concentrations of ovalbumin, or when H-2b B cells were incubated with a hybridoma specific for I-Ab alone. This was interpreted to mean that B cells can be stimulated by reaction of T cells with surface I molecules.

Interleukin-induced increase in Ia expression by normal mouse B cells.

The constitutive culture supernatant (SN) of the macrophage tumor line P388D1 (P388 SN) and the concanavalin A (Con A)-induced culture supernatant of the T cell hybridoma FS6-14.13 (FS6 Con A SN) were shown to contain nonspecific factors capable of inducing increased Ia expression by normal resting B cells in a dose-dependent manner. In six consecutive experiments the relative increase in Ia expression induced by P388 SN was 4.9 +/- 0.9, with FS6 Con A SN 10.7 +/- 1.5, and with a combination of both preparations 13.0 +/- 1.7. This increase in Ia expression was observed to occur in virtually all the B cells, reaching maximum levels within 24 h of culture. The interleukin-induced increase in B cell Ia expression occurred in the absence of ancillary signals provided by ligand-receptor Ig cross-linking and despite the fact that virtually all the control B cells, cultured in the absence of factors, remained in G0. These results suggest that functional receptors for at least some interleukins are expressed on normal resting B cells and their effects can be manifest in the absence of additional activating signals. The increased Ia expression induced by the nonspecific factor preparations was shown to be correlated with enhanced antigen-presenting capacity by the B cells to T cell hybridomas. The nature of the interleukins responsible for these effects remains to be definitively determined, however, the activity of FS6 Con A SN was shown to correlate with B cell growth factor activity and increased B cell Ia expression was not observed using interleukin 2 (IL-2) or interferon-gamma, prepared by recombinant DNA technology.

An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT.

A new tetrazolium salt XTT, sodium 3'-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6- nitro)benzene-sulfonic acid hydrate, was evaluated for use in a colorimetric assay for cell viability and proliferation by normal activated T cells and several cytokine dependent cell lines. Cleavage of XTT by dehydrogenase enzymes of metabolically active cells yields a highly colored formazan product which is water soluble. This feature obviates the need for formazan crystal solubilization prior to absorbance measurements, as required when using other tetrazolium salts such as MTT. Bioreduction of XTT by all the murine cells examined was not particularly efficient, but could be potentiated by addition of electron coupling agents such as phenazine methosulfate (PMS) or menadione (MEN). Optimal concentrations of PMS or MEN were determined for the metabolism of XTT by the T cell lines HT-2 and 11.6, NFS-60 a myeloid leukemia, MC/9 a mast cell line and mitogen activated splenic T cells. When used in combination with PMS, each of these cells generated higher formazan absorbance values with XTT than were observed with MTT. Thus the use of XTT in colorimetric proliferation assays offer significant advantages over MTT, resulting from reduced assay time and sample handling, while offering equivalent sensitivity.

Cyclosporine and FK506 inhibition of murine mast cell cytokine production.

The ability of cyclosporine (CSA) and FK506 to inhibit cytokine production by factor-dependent murine mast cell lines was investigated. The mast cell clone, MC/9, and two mast cell lines, MCIII and MCVI, were stimulated to produce cytokines with phorbol myristate acetate plus the calcium ionophore A23187. The production of cytokines by stimulated mast cells cultured in the presence or absence of drug was monitored by bioassay of culture supernatants for induction of proliferation by factor-dependent cell lines and inhibition of these responses by neutralizing monoclonal antibodies. Both CSA and FK506 inhibited mast cell cytokine production at concentrations comparable to those observed with T cells. However, the degree of inhibition of cytokine production varied among the mast cell lines as well as between different cytokines produced by a given mast cell line. For example, CSA completely inhibited interleukin-2 (IL-2), IL-3, IL-4 and granulocyte-macrophage colony stimulating factor secretion by all three lines, with the exception that IL-2/IL-4 production by MCIII was partially resistant to inhibition by CSA. Similarly, FK506 completely inhibited cytokine production by MC/9, partially inhibited cytokine production by MCIII and had differential effects on IL-3/granulocyte-macrophage colony-stimulating factor and IL-2/IL-4 production by MCVI. Consistent with their ability to selectively inhibit cytokine gene transcription in T cells, neither CSA nor FK506 inhibited factor-dependent proliferation by these mast cell lines. In view of the putative role of cytokines in inflammation and late phase asthmatic reactions, these observations may be of particular significance in development of methods of pharmacologic intervention.

Rapamycin and FK506 differentially inhibit mast cell cytokine production and cytokine-induced proliferation and act as reciprocal antagonists.

We have previously demonstrated that cyclosporine (CSA) and FK506 are able to selectively inhibit cytokine production by murine mast cell lines at concentrations comparable to those observed with thymus-derived lymphocytes (T cells). The selectivity of these effects were demonstrated by the failure of CSA and FK506 to inhibit cytokine-induced mast cell proliferation at equivalent or higher concentrations. In this report, we examined the ability of rapamycin (RAP) to inhibit cytokine production and cytokine-induced proliferation by a factor-dependent murine mast cell line and compared its activity to that of the structurally related macrolide FK506. The mast cell clone, MC/9, was stimulated to produce cytokines with phorbol myristate acetate plus the calcium ionophore A23187, or to proliferate in response to exogenous cytokines such as interleukin-3 and interleukin-4, produced by the helper T cell clone D10.G4. RAP did not inhibit cytokine production by MC/9, even at concentrations greater than 1000 nM. FK506 and CSA inhibited cytokine production with IC50 of 0.8 and 16.2 nM, respectively. In contrast to its lack of effect on cytokine production, RAP potently inhibited cytokine-induced proliferation of MC/9 cells with an IC50 of 1.9 nM. Because RAP and FK506 are structurally related and yet have divergent biological effects, we examined the ability of RAP to antagonize inhibitory effects of FK506 on mast cell cytokine production and the ability of FK506 to antagonize inhibitory effects of RAP on cytokine-induced mast cell proliferation. The addition of RAP in molar excess reversed inhibition of mast cell cytokine production mediated by FK506, but not that of CSA.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorylation and activation of Ca(2+)-sensitive cytosolic phospholipase A2 in MCII mast cells mediated by high-affinity Fc receptor for IgE.

In the present study we examined the activation of Ca(2+)-sensitive cytosolic phospholipase A2 (cPLA2) after aggregation of cell-surface high-affinity Fc receptors for IgE (Fc epsilon RI) on mast cells. MCII mast cells (a factor-dependent bone-marrow-derived murine mast cell line) produce significant amounts of leukotriene C4 (LTC4) (70 ng/10(6) cells) on cross-linking of Fc epsilon RI. Using enzymic and immunochemical analysis we found that cPLA2 is the predominant form of this enzyme in MCII mast cells (0.2 micrograms/mg of total protein) and other forms (i.e. secretory PLA2 or Ca2+ independent cytosolic PLA2) could not be detected. Therefore MCII mast cells represent an excellent cellular model for the study of the biochemical mechanism(s) responsible for Fc epsilon RI-induced activation of cPLA2 and the involvement of cPLA2 in Fc epsilon RI-mediated production of LTC4. After activation of Fc epsilon RI by cross-linking, cPLA2 in MCII mast cells exhibited a decreased electrophoretic mobility and its enzyme activity was increased 3-fold. Treatment with phosphatase reversed both the altered electrophoretic mobility and the enhanced enzyme activity demonstrating that they were the result of Fc epsilon RI-induced phosphorylation. On cross-linking of Fc epsilon RI, cPLA2 was phosphorylated within 30 s and appeared to be an early substrate for Fc epsilon RI-activated protein kinases in MCII mast cells. Tyrosine phosphorylation may be a critical component in this process, as genistein, an inhibitor of protein tyrosine kinases, blocked the activation of cPLA2. Using anti-phosphotyrosine antibodies we observed that the activating phosphorylation was not on tyrosine residues of cPLA2, indicating that tyrosine kinases participate upstream in the signalling cascade that couples Fc epsilon RI to cPLA2. We conclude that in MCII mast cells cPLA2 is activated by kinase-dependent mechanisms and may be responsible for Fc epsilon RI-induced mobilization of arachidonic acid for the generation of LTC4.

Patterns of lymphokine production by primary antigen-specific/MHC restricted murine helper T cell clones.

In this study we examined a panel of CD4+ antigen specific/MHC restricted T cell clones for their ability to secrete IL-2, IL-4, and IFN-gamma upon stimulation with con A, three lymphokines which are diagnostic for the TH1 and TH2 subtypes of helper T cells. Eight of the twelve clones we analyzed did not fit the classical TH1/TH2 patterns of lymphokine secretion. Seven of these clones secreted both IL-2 and IL-4 and two of these also produced IFN-gamma. The remaining non-classical clone secreted IL-4 and IFN-gamma but not IL-2. Data from the subcloning of the IL-2/IL-4/IFN-gamma triple producers were not consistent with the parental lines being a mixture of TH1 and TH2 cells. The IL-2/IL-4 double producers (IFN-gamma negative) cannot be explained by the parental lines being a mixture of the TH1 and TH2 subtypes. Nevertheless, these double producers were subcloned and the results provided convincing evidence that clones which secrete both IL-2 and IL-4 do exist. Lymphokine loss variants involving IL-2, IL-4 or IFN-gamma were observed among subclones derived from the double and triple producers as well as in several parental lines maintained in continuous culture. We also observed the appearance of inducible IFN-gamma production in some subclones derived from parental clones where production of IFN-gamma was not detectable. The phenotypes of these variants failed to indicate an obvious trend toward the TH1 and TH2 subtypes. Thus, our results suggest that more heterogeneity in the population of CD4+ helper T cells exists than can be explained by the TH1 and TH2 subtypes of these cells.

The major histocompatibility complex-restricted antigen receptor on T cells: the genetics of expression of an allotype.

The monoclonal antibody KJ16-133 binds an allelic determinant expressed on the antigen-specific, major histocompatibility complex (MHC)-restricted receptors on approximately 20% of T cells in most mouse strains. The locus controlling the presence or absence of the determinant mapped 9.8 +/- 2.2 centimorgans from the Igk/Ly-2 locus on chromosome 6 in mice, and may be the beta-chain locus. Other genetic loci were identified that controlled the frequency of cells that expressed the allele in positive mice. One of these was the MHC itself, which may control expression of the beta-chain allele by controlling T cell repertoire. The identity of the other, as yet unmapped locus is unknown. KJ16-133 was used to show that T cell receptor gene products are expressed in a manner consistent with allelic exclusion.

Pharmacology of LY315920/S-5920, [[3-(aminooxoacetyl)-2-ethyl-1- (phenylmethyl)-1H-indol-4-yl]oxy] acetate, a potent and selective secretory phospholipase A2 inhibitor: A new class of anti-inflammatory drugs, SPI.

LY315920 is a potent, selective inhibitor of recombinant human, group IIA, nonpancreatic secretory PLA2 (sPLA2). In a chromogenic isolated enzyme assay, LY315920 inhibited sPLA2 activity with an IC50 of 9 +/- 1 nM or 7.3 x 10(-6) mole fraction, which approached the stiochiometric limit of this assay. The true potency of LY315920 was defined using a deoxycholate/phosphatidylcholine assay with a mole fraction of 1.5 x 10(-6). LY315920 was 40-fold less active against human, group IB, pancreatic sPLA2 and was inactive against cytosolic PLA2 and the constitutive and inducible forms of cyclooxygenase. Human sPLA2-induced release of thromboxane A2 (TXA2) from isolated guinea pig lung bronchoalveolar lavage cells was inhibited by LY315920 with an IC50 of 0.79 microM. The release of TXA2 from these cells by N-formyl-methionyl-leucyl-phenylalanine or arachidonic acid was not inhibited. The i.v. administration of LY315920, 5 min before harvesting the bronchoalveolar lavage cells, resulted in the inhibition of sPLA2-induced production of TXA2 with an ED50 of 16.1 mg/kg. Challenge of guinea pig lung pleural strips with sPLA2 produced contractile responses that were suppressed in a concentration-dependent manner by LY315920 with an apparent KB of 83 +/- 14 nM. Contractile responses induced by arachidonic acid were not altered. Intravenous or oral administration of LY315920 to transgenic mice expressing the human sPLA2 protein inhibited serum sPLA2 activity in a dose-related manner over a 4-h time course. LY315920 is a potent and selective sPLA2 inhibitor and represents a new class of anti-inflammatory agent designated SPI. This agent is currently undergoing clinical evaluation and should help to define the role of sPLA2 in various inflammatory disease states.