Omega-3 fatty acids, EPA and DHA induce apoptosis and enhance drug sensitivity in multiple myeloma cells but not in normal peripheral mononuclear cells.

The n-3 polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to enhance the effect of chemotherapeutic drugs in clinical studies in cancer patients and to induce apoptotic tumor cell death in vitro. Until now, EPA and DHA have never been investigated in multiple myeloma (MM). Human myeloma cells (L363, OPM-1, OPM-2 and U266) and normal peripheral blood mononuclear cells were exposed to EPA and DHA, and effects on mitochondrial function and apoptosis, caspase-3 activation, gene expression and drug toxicity were measured. Exposure to EPA and DHA induced apoptosis and increased sensitivity to bortezomib in MM cells. Importantly, they did not affect viability of normal human peripheral mononuclear cells. Messenger RNA expression arrays showed that EPA and DHA modulated genes involved in multiple signaling pathways including nuclear factor (NF) κB, Notch, Hedgehog, oxidative stress and Wnt. EPA and DHA inhibited NFκB activity and induced apoptosis through mitochondrial perturbation and caspase-3 activation. Our study suggests that EPA and DHA induce selective cytotoxic effects in MM and increase sensitivity to bortezomib and calls for further exploration into a potential application of these n-3 polyunsaturated fatty acids in the therapy of MM.

Cigarette smoke suppresses the surface expression of c-kit and FcεRI on mast cells.

Chronic obstructive pulmonary disease (COPD) is a multicomponent disease characterized by emphysema and/or chronic bronchitis. COPD is mostly associated with cigarette smoking. Cigarette smoke contains over 4,700 chemical compounds, including free radicals and LPS (a Toll-Like Receptor 4 agonist) at concentrations which may contribute to the pathogenesis of diseases like COPD. We have previously shown that short-term exposure to cigarette smoke medium (CSM) can stimulate several inflammatory cells via TLR4 and that CSM reduces the degranulation of bone-marrow-derived mast cells (BMMCs). In the current study, the effect of CSM on mast cells maturation and function was investigated. Coculturing of BMMC with CSM during the development of bone marrow progenitor cells suppressed the granularity and the surface expression of c-kit and Fc ε RI receptors. Stimulation with IgE/antigen resulted in decreased degranulation and release of Th1 and Th2 cytokines. The effects of CSM exposure could not be mimicked by the addition of LPS to the culture medium. In conclusion, this study shows that CSM may affect mast cell development and subsequent response to allergic activation in a TLR4-independent manner.

Local free light chain expression is increased in chronic rhinosinusitis with nasal polyps.

BACKGROUND: Free light chain (FLC) concentrations are demonstrated to be increased in different inflammatory disorders and are proposed to mediate mast cell-dependent immune responses. A role for mast cells is suggested in chronic rhinosinusitis with nasal polyposis (CRSwNP), which is characterized by a local Th2 inflammatory response. However, clear mast cell-activating factors are not always apparent. In this study, the presence of FLCs in CRS patients with or without nasal polyps (CRSw/sNP) was investigated and the effect of different treatments on FLC expression was analyzed. METHODS: Nasal tissue, nasal secretion, and serum of control patients, patients with CRSwNP, and CRSsNP were analyzed for the presence of kappa and lambda FLC. The expression of FLCs in nasal polyp tissue was investigated using immunohistochemistry. In addition, FLC was measured in serum and nasal secretion of nasal polyp patients treated with methylprednisolone, doxycycline, anti-IL-5, or placebo. RESULTS: Free light chain concentrations were increased in nasal secretion and mucosal tissue homogenates in patients with chronic rhinosinusitis, and this effect was most prominent in CRSwNP patients. Immunohistochemical analysis confirmed the increased FLC concentrations in nasal polyp tissue. In CRSwNP patients, treatment with methylprednisolone or anti-IL-5 resulted in the reduction in systemic or local FLC concentrations, respectively. CONCLUSION: The presence of FLC in CRSwNP and CRSsNP suggests a possible role in mediating the local immune reaction in the paranasal cavities. Furthermore, the decrease in local FLCs after treatment with anti-IL-5 presumes that IL-5 creates an environment that favors FLC production.

Decrease in immunoglobulin free light chains in patients with rheumatoid arthritis upon rituximab (anti-CD20) treatment correlates with decrease in disease activity.

OBJECTIVES: Immunoglobulin (Ig) free light chains (FLCs) are short-lived B cell products that contribute to inflammation in several experimental disease models. In this study, FLC concentrations in inflamed joints of patients with rheumatoid arthritis (RA) as compared to patients with osteoarthritis were investigated. In addition, the relationship of FLCs and disease activity upon B cell depletion (rituximab) in patients with RA was studied. METHODS: Synovial fluid (SF) and tissue from patients with RA were analysed for local presence of FLCs using ELISA and immunohistochemistry. In addition, FLC concentrations were measured (at baseline, 3 and 6 months after treatment) in 50 patients with RA with active disease who were treated with rituximab. Changes in FLCs were correlated to changes in disease activity and compared to alterations in IgM, IgG, IgA, IgM-rheumatoid factor (RF) and IgG-anti-citrullinated protein antibody (ACPA) concentrations. RESULTS: FLCs were detected in synovial tissue from patients with RA, and high FLC concentrations were found in SF from inflamed joints, which positively correlate with serum FLC concentrations. Serum FLC concentrations significantly correlated with disease activity score using 28 joint counts, erythrocyte sedimentation rate (ESR) and C reactive protein, and changes in FLC correlated with clinical improvement after rituximab treatment. Moreover, effect of treatment on FLC concentrations discriminated clinical responders from non-responders, whereas IgM-RF and IgG-ACPA significantly decreased in both patient groups. CONCLUSIONS: FLCs are abundantly present in inflamed joints and FLC levels correlate with disease activity. The correlation of FLC concentrations and disease activity indicates that FLCs may be relevant biomarkers for treatment response to rituximab in patients with RA and suggests that targeting FLC may be of importance in the therapy of RA.

Depletion of CD4+CD25+ T cells switches the whey-allergic response from immunoglobulin E- to immunoglobulin free light chain-dependent.

BACKGROUND: Symptoms of allergy are largely attributed to an IgE-mediated hypersensitivity response. However, a considerable number of patients also exhibit clinical features of allergy without detectable systemic IgE. Previous work showed that Ig-free light chains (IgLC) may act as an alternate mechanism to induce allergic responses. CD4+CD25+ T cells are crucial in the initiation and regulation of allergic responses and compromised function might affect the response to allergens. OBJECTIVE: To examine the contribution of CD4+CD25+ T cells and IgLC towards the whey-allergic response. METHODS: Mice were sensitized orally with whey using cholera toxin as an adjuvant. CD25+ T cells were depleted in vivo using a CD25 mAb. The acute allergic skin response to whey and ex vivo colon reactivity was measured in the presence or absence of F991, a specific inhibitor of IgLC. Serum whey-specific antibodies and IgLC in serum and mesenteric lymph node (MLN) supernatants were measured. Depletion of CD4+CD25+ T cells was confirmed in the spleen. RESULTS: Anti-CD25 treatment strongly reduced whey-specific antibody levels and resulted in a partial depletion of effector T cells and a major depletion of Foxp3(+) regulatory T cells. Surprisingly, despite the abolished specific IgE response, the acute allergic skin response to whey was not affected. IgLC levels were enhanced in the serum and MLN supernatants of CD25-depleted sensitized mice. F991 inhibited the acute skin response and colon hyperreactivity in anti-CD25-treated mice, indicating that these responses were mainly IgLC dependent. CONCLUSIONS: Depletion of CD4+CD25+ T cells resulted in a switch from an IgE- to an IgLC-dependent acute skin response and functional hyperresponsiveness of the colon. Our data suggest that CD25+ T cells play a crucial role in balancing cow's milk allergy between IgE and IgE-independent responses and both mechanisms might play a role in allergic responses to the same allergen.

Cigarette smoke suppresses in vitro allergic activation of mouse mast cells.

BACKGROUND: Mast cells are important effector cells in innate or acquired immunity that contribute to host defence. Excessive activation of mast cells can result in the development of allergic diseases, including atopic asthma. Mast cell activation by IgE and specific antigen induces the cells to release spasmogenic, vasoactive and pro-inflammatory mediators, which enhance airway smooth muscle contraction, vascular permeability and inflammatory cell recruitment. Recently, we have demonstrated that exposure of mast cells to cigarette smoke medium (CSM) triggered mast cells to produce chemokines. On the other hand, smoking may decrease the risk of allergic sensitization, which could be explained by a reduced IgE production or a diminished response of mast cells to activation of the IgE receptor. OBJECTIVE: In this study, we investigated the effect of CSM on the allergic activation of mast cells through IgE and antigen. METHODS: Primary cultured murine mast cells were exposed to CSM and activated with IgE and antigen or lipopolysaccharide (LPS). The release of granules, production of leukotrienes, chemokines and cytokines was determined in the supernatants by ELISA. The effect of CSM exposure on intracellular signalling, especially the nuclear factor (NF)-kappaB and extracellular signal-regulated kinase (Erk)1/2 pathways, was analysed by Western blotting. RESULTS: CSM suppressed IgE-mediated degranulation and cytokine release, but no effect was observed on leukotriene release. CSM induced phosphorylation of Erk1/2 in mast cells. In CSM-exposed mast cells, activating transcription factor (ATF)-1 was phosphorylated after stimulation with IgE/Ag. LPS-activated mast cells were not influenced by CSM. CONCLUSION: Our study suggests that exposure to cigarette smoke may lead to a reduced allergic activation of mast cells without affecting their response to activation via e.g. bacterial-derived LPS.

Atopic and non-atopic allergic disorders: current insights into the possible involvement of free immunoglobulin light chains.

Allergic diseases have become a serious global health problem in the developed world. IgE interacting with its high-affinitiy receptor FcepsilonRI is considered a major contributing factor to most types of allergies, but depending on the type of allergy, however, a subgroup of patients displays common symptoms and yet lack elevated levels of total serum IgE and/or antigen-specific IgE. Novel therapeutic strategies such as anti-IgE therapy may therefore not be applicable to these patients. It is clear, however, that these patients do display activation of mast cells. In several patients suffering from immunological disorders, an increase in free immunoglobulin (IG) light chain levels can be detected. Previously, we have described the capability of free light chains to elicit immediate hypersensitivity responses. In this Opinion article, we will discuss the role of IgE- and non-IgE-mediated mechanisms in allergic disorders and point out a possible role of free IG light chains in the pathogenesis of the non-atopic types of these allergies.

Topical application of F991, an immunoglobulin free light chain antagonist, prevents development of contact sensitivity in mice.

BACKGROUND: Exposure to reactive chemicals or environmental allergens can lead to hypersensitivity reactions in the skin of predisposed people. Most of these reactions are of atopic origin, but a subgroup of patients exhibits skin hypersensitivity reactions without features of atopy. OBJECTIVE: This study was undertaken to examine the effect of inhibiting the action of Ig-free light chains in a murine model for non-atopic skin hypersensitivity by dermal application of the free light chain antagonist F991. METHODS: To study the efficacy of F991, BALB/c mice were either passively immunized with trinitrophenyl (TNP)-specific immunoglobulin light chains (IgLC) and challenged with the hapten picryl chloride (PCl) or actively skin-sensitized and challenged with dinitrofluorobenzene (DNFB). The effect of F991 or control treatment was investigated by measuring local edema formation and the production of pro-inflammatory cytokines. RESULTS: Passive immunization with TNP-specific IgLC resulted in an increase in ear swelling 2 h after PCl challenge. F991 inhibited this enhanced ear swelling in a dose-dependent manner when applied 4 h before the sensitization with IgLC. F991 also inhibited DNFB-induced contact hypersensitivity reaction in the mouse skin 2 and 24 h after challenge when applied before challenge. Besides the prophylactic action, F991 when applied 2 h after DNFB-challenge, it was also able to attenuate symptoms of the DNFB-induced hypersensitivity reaction at 24 h after challenge. We showed that the beneficial effects of F991 are restricted to the side of application. CONCLUSION: F991 is able to effectively alleviate symptoms of contact sensitivity in mice. Our study suggests that local interference with IgLC-induced allergic symptoms may be attractive in the treatment of hypersensitivity responses.

Peptoid - peptide hybrids that bind Syk SH2 domains involved in signal transduction.

Peptoid-peptide hybrids are oligomeric peptidomimetics that contain one or more N-substituted glycine residues. In these hybrids, the side chains of one or several amino acids are "shifted" from the alpha-carbon atom to the amide nitrogen atom. A library of phosphorylated peptoid-peptide hybrids derived from the sequence pTyr-Glu-Thr-Leu was synthesized and tested for binding to the tandem SH2 domain of the protein tyrosine kinase Syk. A considerable influence of the side chain position was observed. Compounds 19-21, 24, and 25 comprising a peptoid NpTyr and/or NGlu residue did not show any binding. Compounds 22, 23, and 26 containing an NhThr (hThr=homothreonine) and/or NLeu peptoid residue showed binding with IC(50) values that were only five to eight times higher than that of the tetrapeptide lead compound 18. These data show that side chain shifting is possible with retention of binding capacity, but only at the two C-terminal residues of the tetramer. This method of a peptoid scan using peptoid-peptide hybrids appears to be very useful to explore to what extent a peptide sequence can be transformed into a peptoid while retaining its affinity.

Stem cell factor and interleukin-4 increase responsiveness of mast cells to substance P.

The response of mast cells (MC) to non-IgE-mediated stimulation is critically dependent on the population of MC examined. The neuropeptide Substance P (SP) has been reported to activate connective tissue-type MC (CTMC), while mucosal MC (MMC) are not activated by SP. We examined the effect of stem cell factor (SCF) plus interleukin-4 (IL-4) on SP-initiated activation of bone marrow-derived MC (BMMC). Mouse MC, derived from a culture of BM cells with IL-3, were subsequently treated with recombinant SCF plus IL-4 for 6 days. Responsiveness to SP was monitored measuring beta-hexosaminidase and lipid mediator release. Histochemical staining, histamine analysis, and granule protease expression were achieved to characterize the cells. In contrast to IL-3 grown cells, SCF/IL-4-exposed cells showed functional responsiveness to release beta-hexosaminidase (42.25% +/- 1.46% at SP concentration of 100 microM) and produce leukotriene C(4) (LTC(4)) (7.4 +/- 1.5 ng/10(6) cells)/prostaglandin D(2) (PGD(2)) (2.0 +/- 0.3 ng/10(6) cells) upon stimulation by SP. The increase in sensitivity of the cells to SP was not due to differentiation into CTMC, as the cells remained heparin negative. Both SCF and IL-4 were needed because SCF or IL-4 alone were insufficient to keep cells viable after 3 to 4 days post coculture. SP-induced secretion from BMMC cultured in medium containing SCF plus IL-4 (25.76% +/- 1.83%) was higher in comparison with cells cultured with SCF plus IL-3 (8.85% +/- 0.68%).These findings indicate that temporal changes in cytokine expression can influence the sensitivity of MC to non-immunologic stimuli. Local cytokine production leading to an increase in MC responsiveness to SP and inducing secretion of granule content and lipid generation may, therefore, propagate and worsen inflammatory conditions.

Ecto-protein kinases: ecto-domain phosphorylation as a novel target for pharmacological manipulation?

An increasing number of studies document the presence of protein kinases facing outwards at the cell surface of a diverse array of cells. These ecto-protein kinases phosphorylate cell-surface proteins and soluble extracellular substrates, and thus could affect many physiological processes involving cell-cell contacts, cellular differentiation and proliferation, ion fluxes and cellular activation. To date, only limited attention has been paid to exploring ecto-protein kinases as possible pharmacological targets. Here, the identification and physiological role of ecto-protein kinases in different biological systems is described; it is suggested that ecto-protein kinases are attractive and novel candidates for pharmacological manipulation under various (patho)physiological conditions.

Stem cell factor and interleukin-4 induce murine bone marrow cells to develop into mast cells with connective tissue type characteristics in vitro.

In this study, we have developed a method to obtain mast cells with connective tissue type mast cell (CTMC) characteristics directly from mouse bone marrow (BM) cells. BM cells were grown for 3 weeks in presence of interleukin-4 (IL-4) plus stem cell factor (SCF). SCF alone poorly supported growth and development of mast cells. IL-4 dose-dependently enhanced the expression of c-kit and high-affinity receptor for IgE (Fc(epsilon)RI) on the cell surface of SCF-cultured BM cells. Furthermore, cytoplasmic granulation and histamine synthesis of BM-derived mast cells were increased in presence of IL-4 and SCF. Histochemical staining demonstrated that granules were safranin positive. BM-derived mast cells could be activated for granule exocytosis (beta-hexosaminidase release) and lipid mediator generation (LTC4 production) via Fc(epsilon)RI after sensitization with IgE and subsequent crosslinking with multivalent antigen. In addition, mast cells derived from BM cells cultured with SCF plus IL-4 could be activated by substance P, a nonimmunologic stimulus, to release beta-hexosaminidase. The results presented indicate that IL-4 and SCF both have a prominent role in the development of mast cells from murine BM cells in vitro. Mast cells can directly be derived from BM cells in presence of SCF and IL-4 and the cultured cells show typical hallmarks of CTMC, indicating that precursor cells for CTMC may be present in BM. The described culture procedure may be useful to investigate the molecular aspects of the development of committed mast cell lineages.

Phosphorylation of T-lymphocyte plasma membrane-associated proteins by ectoprotein kinases: implications for a possible role for ectophosphorylation in T-cell effector functions.

Extracellular adenosine triphosphate (ATPo) has been suggested to play a role in lymphocyte effector functions. Recently, it has been suggested that MgATP2- may be the molecular species which is involved in modulating the lytic interaction between cytotoxic T-lymphocytes (CTL) and their target cells. In this study, we provide evidence that ATPo mediates the phosphorylation of extracellular proteins on T-lymphocytes through the action of ectoprotein kinases. The ectophosphorylation is temperature-dependent, supported by Mg2+ and Mn2+, and both ATP and GTP, whereas kinase activity and/or substrates were removed by pretreatment of intact lymphocytes with trypsin. We show the presence of extracellular ATP/GTP-binding sites, indicating the presence of ectoenzymes on intact lymphocytes. The major ectoprotein kinase was identified as a casein kinase II-like protein kinase and could be inhibited by heparin, whereas its activity was enhanced by spermine. The ectoprotein kinase showed remarkable substrate specificity, phosphorylating the serum protein vitronectin, but not fibronectin. In experiments with the cell-impermeable protein kinase inhibitor K-252b, we demonstrate the possible functional importance of ectoprotein kinase in CTL-mediated cytotoxicity, i.e., target cell death was completely blocked by K-252b without affecting intracellular phosphorylation. These results suggest that ectoprotein phosphorylation may possibly be an important event in immunologically relevant cell-cell interactions.

Nitric oxide production by macrophages stimulated by antigen-binding T-cell factors.

Contact sensitivity to small molecular weight compounds is accompanied by the production of antigen-specific T-cell factors (TCF) shortly after skin application of the sensitizing agents. In this study, we show that macrophages can be activated by these TCF to generate large amounts of nitric oxide (NO). Incubation of the murine macrophage cell line J774 for 24 h with TCF raised against dinitrofluorobenzene (DNFB) or picryl chloride (PCL) resulted in a nitrite accumulation in the culture medium. Priming of J774 with rIFN-gamma synergistically enhanced stimulation of NO synthesis by DNFB-F and PCL-F. A possible contribution of lipopolysaccharide (LPS) as a contaminant of the TCF was excluded. The enhanced production of NO after stimulation with TCF was accompanied with an increased expression of inducible NO synthase. Inclusion of inhibitors of protein tyrosine kinase and protein kinase C inhibited the TCF-induced NO production by macrophages, indicating the involvement of both protein kinases in the signaling pathway activated by TCF. Since NO is an important biological mediator with many immunoregulatory properties, our results suggest a potential role for increased NO production by macrophages in the elicitation of contact sensitivity to small molecular weight compounds.

LFA-1, and not Mac-1, is crucial for the development of hyperreactivity in a murine model of nonallergic asthma.

In this study, we investigated the importance of the beta 2-integrins for the development of tracheal hyperreactivity in a murine model for nonallergic asthma. The response was induced by skin sensitization with dinitrofluorobenzene (DNFB) followed by an intranasal challenge with the same hapten. Twenty-four hours after the challenge, tracheal hyperreactivity, a decrease in T cells in the blood, and increased neutrophil numbers in bronchoalveolar lavage fluid (BALF) and blood were observed. Monoclonal antibodies (mAbs) directed against the alpha-chains of LFA-1 (FD441.8) and Mac-1 (M1/70) were injected intravenously 2 h before and 2 h after the challenge. Treatment with anti-LFA-1 mAb totally inhibited the development of tracheal hyperreactivity measured 24 h after the challenge, whereas anti-Mac-1 mAb had only a partial effect on this response. The decrease in T cells in the blood, which was also evident 24 h after the challenge, was totally inhibited by treatment with anti-LFA-1, whereas anti-Mac-1 had little effect. The increase in the number of neutrophils in BALF at this time point was completely inhibited by both anti-LFA-1 and anti-Mac-1. In summary, evidence presented in this report highlights the possible importance of the adhesion molecule LFA-1 in the development of tracheal hyperreactivity. Our results suggest that LFA-1 present on T cells may play an integral role in this response.

Depletion of ATP but not of GSH affects viability of rat hepatocytes.

The purpose of this study was to examine the role of glutathione depletion and alterations in the energy status in the induction of acute cytotoxicity to freshly isolated rat hepatocytes. Depletion of intracellular glutathione by diethyl maleate and phorone to levels below 5% of control did not induce loss of viability nor loss of intracellular ATP. Ethacrynic acid, a compound known to deplete mitochondrial GSH in addition to cytosolic GSH, induced cell killing after a depletion of ATP, next to GSH depletion. The results confirmed that depletion of intracellular glutathione alone does not necessarily result in cell killing. Only when glutathione depletion is succeeded by reduction in ATP levels, loss of cell viability is observed. The relationship between alterations in the energy status and the induction of cell death was further substantiated by inhibition of glycolytic and mitochondrial ATP generation. Treatment of hepatocytes either with iodoacetic acid to inhibit glycolysis (in hepatocytes from fed rats) or with potassium cyanide to inhibit mitochondrial respiration (in hepatocytes from both fed and fasted rats) revealed that depletion of intracellular ATP could lead to lethal cell injury. The susceptibility of cells to metabolic inhibition was better reflected by the rate of reduction in the energy charge than by the reduction of ATP alone. In conclusion, our results suggest that alterations of the energy status may be a critical event in the induction of irreversible cell injury. Depletion of cellular GSH is only cytotoxic when followed by a reduction of the energy charge.

Poly-(ADP-ribose) polymerase partially contributes to target cell death triggered by cytolytic T lymphocytes.

We hypothesized that CTL-induced target cell (TC) death is partially due to processes that follow the DNA damage in target cells and include the activation of poly-ADP-ribose transferase (PADPRT) by DNA strand breaks. According to this model, the activated PADPRT is expected to deplete NAD, ATP, and to contribute to the TC death. We used inhibitors of PADPRT and a PADPRT-deficient cell mutant, as well as other nucleated TC and SRBC to test the role of PADPRT in CTL-induced cytotoxicity. It is found that inhibitors of PADPRT (3-aminobenzamide, benzamide (aromatic amides)) and nicotinamide all inhibit the CTL-mediated lysis of both Ag-specific TC and of Ag-nonbearing TC. The effect of PADPRT inhibitors was not due to inhibition of the lethal hit delivery by CTL, because in parallel control experiments, the same inhibitors did not interfere with CTL-induced lysis of SRBC, cells that are devoid of nuclei and PADPRT. Moreover, the effect of inhibitors of PADPRT did not affect earlier stages of lethal hit delivery because 3-aminobenzamide and benzamide did not interfere with CTL-induced DNA fragmentation in TC at concentration which protected TC lysis. Importantly, a PADPRT-deficient cell line was also much more resistant to CTL-induced lysis as tested in retargeting (4 and 8 h) assays; this was expected if activation of PADPRT is indeed involved in TC death. Control experiments reveal that the relative resistance of the PADPRT-deficient cell mutant to CTL-induced lysis was not related to its impaired ability to form conjugates and to trigger CTL (as tested in granule exocytosis assay). In addition, PADPRT-deficient cells were as susceptible to CTL-induced DNA fragmentation as were the control cells; yet, they were resistant to CTL-induced 51Cr-release. Control cells and PADPRT-deficient mutant were equally susceptible to antibody+C'-mediated lysis. Our data support the view that the activation of PADPRT can contribute to the CTL-induced cytolysis of some TC, but is not involved in lysis of other TC, as evidenced by the ability of CTL to efficiently lyse SRBC. These data suggest that there could be multiple molecular pathways of TC death in CTL-mediated cytotoxicity and the relative contribution of PADPRT and/or other enzymes will reflect the individual make-up of a particular TC.

Modulation of cytolytic T lymphocyte functions by an inhibitor of serine/threonine phosphatase, okadaic acid. Enhancement of cytolytic T lymphocyte-mediated cytotoxicity.

A specific and potent inhibitor of protein phosphatases 1 and 2A, okadaic acid (OA), and its inactive analog, tetramethyl ether (OA-TME), were tested in the cytotoxicity and granule exocytosis assays of CTL activation. At low concentrations OA enhanced, whereas at higher concentrations OA inhibited, CTL responses. The Ag-specific and retargeted cytotoxicity, granule exocytosis induced by target cell (TC), anti-TCR mAb, or PMA and A23187, and conjugate formation with TC were inhibited by pretreatment of CTL with OA as expected if protein phosphatases and protein dephosphorylation were indeed involved in the TCR-mediated signal transduction and effector responses of CTL. Cytotoxicity and granule exocytosis were unaffected by pretreatment of CTL with OA-TME. The inhibitory effect of OA on the exocytic response of CTL induced by TC and anti-TCR mAb can be dissociated from the inhibition of the response to PMA and A23187, suggesting the involvement of a serine and/or threonine protein phosphatase in the early events of transmembrane signaling. At lower concentrations, OA, but not OA-TME, was able to enhance the Ag-specific cytotoxicity and TC-induced exocytosis from CTL clones. The enhancement of these TCR-mediated responses of CTL was observed only if the activation was induced by the Ag on the TC surface, because OA did not enhance either the anti-TCR mAb-induced exocytosis of granules from the CTL clone or lysis of the Ag-nonbearing TC by CTL in a retargeting assay. The biphasic character of the effects of OA on CTL-TC interactions suggests the existence of at least two functionally distinct phosphatases in CTL. The ability of OA to enhance the Ag-specific response is unique and indicates the presence of an inhibitory phosphoprotein phosphatase that should be considered as a participant in the down-regulation of the cell-cell interactions between CTL and TC. The inhibitory effects of OA on both TC-induced and anti-TCR mAb-triggered CTL responses at higher concentrations point to the importance of yet another phosphatase in the CTL-TC interactions and in the TCR-mediated transmembrane signaling. The use of OA may help to decipher the details of biochemical changes involved in T lymphocyte effector functions.