Mechanism of glutamine inhibition of cytosolic phospholipase a2 (cPLA2 ): Evidence of physical interaction between glutamine-Induced mitogen-activated protein kinase phosphatase-1 and cPLA2.

Non-essential amino acid L-glutamine (Gln) possesses anti-inflammatory activity via deactivating cytosolic phospholipase A2 (cPLA2 ). We showed previously that Gln deactivated cPLA2 indirectly via dephosphorylating p38 mitogen-activated protein kinase (MAPK), the major kinase for cPLA2 phosphorylation, through inducing MAPK phosphatase-1 (MKP-1). In this study, we investigated the precise mechanism underlying Gln deactivation of cPLA2 . In lipopolysaccharide (LPS)-treated mice, Gln injection resulted in dephosphorylation of phosphorylated cPLA2 (p-cPLA2 ), which coincided with rapid Gln induction of MKP-1. MKP-1 small interfering RNA (siRNA) abrogated the ability of Gln to induce MKP-1 as well as the dephosphorylation of cPLA2 . Co-immunoprecipitation and in-situ proximity ligation assay revealed a physical interaction between MKP-1 and p-cPLA2 . In a murine model of allergic asthma, we also demonstrated the physical interaction between MKP-1 and p-cPLA2 . Furthermore, Gln suppressed various allergic asthma phenotypes, such as neutrophil and eosinophil recruitments into the airway, airway levels of T helper type 2 (Th2) cytokines [interleukin (IL)-4, IL-5 and IL-13], airway hyperresponsiveness, mucin production and metabolites (leukotriene B4 and platelet-activating factor) through inhibiting cPLA2 in a MKP-1-dependent manner. These data suggest that MKP-1 uses cPLA2 , in addition to p38, as a substrate, which further potentiates the anti-inflammatory action of Gln.

Glutamine preferentially inhibits T-helper type 2 cell-mediated airway inflammation and late airway hyperresponsiveness through the inhibition of cytosolic phospholipase A(2) activity in a murine asthma model.

BACKGROUND: The non-essential amino acid, l-glutamine (Gln), is abundant in the human body. Gln exhibits beneficial effects on endotoxic shock through the inhibition of cytosolic phospholipase A(2) (cPLA(2)) activity. cPLA(2) has been reported to be implicated in the pathogenesis of asthma, but the effects of Gln on asthma have not yet been defined. OBJECTIVE: To investigate the effects of Gln on allergic bronchial inflammation and airway hyperresponsiveness (AHR), and to determine the possible action mechanisms of Gln in a murine model of asthma. METHODS: cPLA(2) phosphorylation was assessed by immunoprecipitation and Western blotting. Smears of bronchoalveolar lavage cells were stained with Diff-Quik solution for differential cell counting. Airway levels of the proteins [T-helper type-1 (Th1) and Th2 cytokines, and mucin] were measured by ELISA. mRNA expression of cytokines was assessed by real-time RT-PCR. AHR was assessed as a change in airway resistance (RL). Histological studies were performed to assess the levels of mucin and pulmonary inflammation. RESULTS: Systemic Gln administration inhibited cPLA(2) phosphorylation and its enzymatic activity in the lungs. Additionally, Gln effectively suppressed the key features of Th2-dependent asthmatic features, such as airway eosinophilia, mucus formation, and airway type 2 cytokine production, as well as late AHR. CONCLUSION: Gln was found to be effective in the suppression of Th2-dependent phenotypes and late AHR, and this effect of Gln appeared to be at least partially attributable to its ability to suppress cLPA(2) activity in the airway. Our results suggest that clinical use of Gln for patients with asthma may be beneficial.

Platelet-activating factor-induced early activation of NF-kappa B plays a crucial role for organ clearance of Candida albicans.

In this study, we have investigated the mechanisms underlying organ susceptibility to candida infection. Infection of BALB/c mice with Candida albicans led to both an early (1-8 h) and late (24-48 h) activation of NF-kappaB in the organs resistant to C. albicans, including the lung and spleen. In susceptible organs such as the kidneys, early activation of NF-kappaB was not observed. The kinetics of TNF-alpha mRNA expression paralleled those of NF-kappaB activation in all organs examined. Blocking the effects of endogenous platelet-activating factor (PAF) by pretreatment with the PAF antagonist BN50739 or antioxidants significantly reduced the early activity of NF-kappaB and TNF-alpha mRNA expression, and increased the recovery of C. albicans in the lung and spleen. Importantly, administration of PAF 5 min prior to the infection resulted in the appearance of early activities of NF-kappaB and TNF-alpha mRNA expression, followed by a nearly complete clearance of the organisms in the kidneys. Pretreatment with anti-TNF-alpha Ab resulted in an enhanced susceptibility to C. albicans, and the PAF-mediated resistance was abrogated by anti-TNF-alpha in all organs examined. These data indicated that endogenously produced PAF in response to C. albicans is a key molecule involved in the early activation of NF-kappaB, which, in turn, renders the organ resistant to the fungus by promoting the production of anti-candidal proinflammatory cytokines such as TNF-alpha. Susceptible organs, including the kidneys, lack the capacity to generate a sufficient PAF-induced early NF-kappaB response.

Selective involvement of reactive oxygen intermediates in platelet-activating factor-mediated activation of NF-kappaB.

Although it has been suggested that some biological activities of platelet-activating factor (PAF) are mediated by, at least in part, reactive oxygen intermediates (ROI), the precise mechanisms underlying the interaction between the two remains to be elucidated. Antioxidants, such as alpha-tocopherol acid succinate, N-acetyl-L-Cysteine, pyrrolidinedithiocarbamate failed to inhibit PAF-induced immediate systemic reactions such as lethality, symptoms of disseminated intravascular coagulation, and histological changes such as pulmonary edema and hemorrhage in renal medullae 10 min following PAF injection. In contrast. antioxidants significantly inhibited both the in vivo and in vitro PAF-induced NF-kappaB activation and NF-kappaB-dependent TNF-alpha expression. The effects of the antioxidants were due to their inhibition of PAF-induced degradation of IkappaBalpha, a protein responsible for keeping NF-kappaB in an inactive form. A protein tyrosine kinase and N-tosyl-L-phenylalanine chloromethyl ketone sensitive serine protease were involved in both PAF- and H2O2-induced NF-kappaB activation. Collectively, these data indicate that the PAF-induced NF-kappaB activation is selectively mediated through the generation of ROI.

A subclass of Ras proteins that regulate the degradation of IkappaB.

Small guanosine triphosphatases, typified by the mammalian Ras proteins, play major roles in the regulation of numerous cellular pathways. A subclass of evolutionarily conserved Ras-like proteins was identified, members of which differ from other Ras proteins in containing amino acids at positions 12 and 61 that are similar to those present in the oncogenic forms of Ras. These proteins, kappaB-Ras1 and kappaB-Ras2, interact with the PEST domains of IkappaBalpha and IkappaBbeta [inhibitors of the transcription factor nuclear factor kappa B (NF-kappaB)] and decrease their rate of degradation. In cells, kappaB-Ras proteins are associated only with NF-kappaB:IkappaBbeta complexes and therefore may provide an explanation for the slower rate of degradation of IkappaBbeta compared with IkappaBalpha.

Chloromethyl ketones inhibit interleukin-12 production in mouse macrophages stimulated with lipopolysaccharide.

Interleukin-12 (IL-12) plays a pivotal role in the development of T-helper type 1 (Th1) immune response, which may be involved in the pathogenesis of chronic inflammatory autoimmune disorders. In this study, we investigated the effects of N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK), serine protease inhibitors, on the production of IL-12 from macrophages stimulated with lipopolysaccharide (LPS). TPCK and TLCK potently inhibited this LPS-induced IL-12 production in a dose-dependent manner. The effect of TPCK and TLCK on the IL-12 p40 promoter activation was analyzed by transfecting monocytic RAW264.7 cells with p40 promoter-reporter constructs. The repressive effect maps to a region in the p40 promoter containing a binding site for NFkappaB (p40-kappaB). A linker scan mutant of the p40-kappaB site abrogates the inhibitory effect on the p40 promoter, confirming the functional relevance of the NFkappaB site. Our results show that TPCK and TLCK inhibit NFkappaB-mediated IL-12 production in macrophages. reserved.

Sulfasalazine prevents T-helper 1 immune response by suppressing interleukin-12 production in macrophages.

Interleukin-12 (IL-12) plays a pivotal role in the development of T-helper 1 (Th1) immune response, which may be involved in the pathogenesis of chronic inflammatory autoimmune disorders. In this study we investigated the effects of sulfasalazine, a drug for treating inflammatory bowel disease and rheumatoid arthritis, on the production of IL-12 from mouse macrophages stimulated with lipopolysaccharide (LPS). Sulfasalazine potently inhibited the production of IL-12 in a dose-dependent manner, in part through the down-regulation of nuclear factor kappaB (NFkappaB) activation in IL-12 p40 gene. Activation of macrophages by LPS resulted in markedly enhanced binding activities to the kappaB site, which significantly decreased upon addition of sulfasalazine as demonstrated by an electrophoretic gel shift assay. Importantly, macrophages pretreated with sulfasalazine either in vitro or in vivo reduced their ability to induce interferon-gamma (IFN-gamma) and increased the ability to induce IL-4 in antigen-primed CD4+ T cells. From these results, sulfasalazine may induce the Th2 cytokine profile in CD4+ T cells by suppressing IL-12 production in macrophages, and sulfasalazine-induced inhibition of IL-12 production in macrophages may explain some of the known biological effects of sulfasalazine.

Preparation of dopamine transporter-specific antibodies using molecular cloned genes.

Dopamine transporter (DAT) plays the most important role in terminating the actions of dopamines released into the synaptic cleft. DAT is also the target of various psychotropic drugs such as cocaine and amphetamine. In this study we prepared DAT-specific antibodies using the 2nd extracellular loop of rat DAT as an antigen. The 2nd extracellular loop of the rat DAT was expressed in bacteria as a fusion protein with glutathione-S-transferase, and injected into rabbits to raise antibodies. Produced antibodies clearly recognized the rat DAT in ELISA, immunoblotting, and immunoprecipitation. As expected from the high sequence homology between the rat and human DAT, the antibodies raised for the rat DAT cross-reacted with the human DAT in the immunoblotting. Considering the specificity for DAT with wide range of applications such as ELISA, immunoblotting, and immunoprecipitation, these antibodies would be valuable tool for understanding the pharmacological actions of dopamine transporter and drug addiction.

Glucocorticoids prevent NF-kappaB activation by inhibiting the early release of platelet-activating factor in response to lipopolysaccharide.

Lipopolysaccharide (LPS) is a known inducer of numerous pro-inflammatory events including the production of platelet-activating factor (PAF). PAF released in response to LPS is a major contributor to the pathological events associated with endotoxemia. The present study demonstrates that dexmethasone (DEX) inhibited the LPS-induced early plasma PAF raise in a dose- and time-dependent manner. In addition, DEX prevented the subsequent PAF-mediated pathological phenomena such as anaphylactic shock-like symptoms, symptoms of disseminated intravascular coagulation and hemorrhage in renal medullae. DEX or the PAF antagonist BN 50739 significantly inhibited LPS-induced NF-kappaB activation. The inhibition of NF-kappaB activation by DEX was overcome by the injection of exogenous PAF. Administration of PAF or LPS resulted in a rapid loss of IkappaBalpha protein. The LPS-induced degradation of IkappaBalpha was prevented by pretreatment with BN 50739, suggesting that PAF is a critical intermediate in the LPS-triggered degradation of IkappaBalpha protein. DEX prevented the LPS-induced IkappaBalpha degradation, which was also reversed by exogenous PAF. Administration of DEX or BN 50739 caused an increase in cytoplasmic IkappaBalpha level. Our results indicate that DEX inhibits IkappaBalpha degradation and subsequent NF-kappaB activation through blocking the initial release of PAF.

Retinoids inhibit interleukin-12 production in macrophages through physical associations of retinoid X receptor and NFkappaB.

Lipopolysaccharide (LPS) increases the production of interleukin-12 (IL-12) from mouse macrophages via a kappaB site within the IL-12 p40 promoter. In this study, we found that retinoids inhibit this LPS-stimulated production of IL-12 in a dose-dependent manner. The NFkappaB components p50 and p65 bound retinoid X receptor (RXR) in a ligand-independent manner in vitro, and the interaction interfaces involved the p50 residues 1-245, the p65 residues 194-441, and the N-terminal A/B/C domains of RXR. Activation of macrophages by LPS resulted in markedly enhanced binding activities to the kappaB site, which significantly decreased upon addition of retinoids, as demonstrated by the electrophoretic mobility shift assays. In cotransfections of CV-1 and HeLa cells, RXR also inhibited the NFkappaB transactivation in a ligand-dependent manner, whereas a mutant RXR lacking the AF2 transactivation domain, which serves as ligand-dependent binding sites for transcription integrators SRC-1 and p300, was without any effect. In addition, coexpression of increasing amounts of SRC-1 or p300 relieved the retinoid-mediated inhibition of the NFkappaB transactivation. From these results, we propose that retinoid-mediated suppression of the IL-12 production from LPS-activated macrophages may involve both inhibition of the NFkappaB-DNA interactions and competitive recruitment of transcription integrators between NFkappaB and RXR.

Immunoglobulin E-dependent active fatal anaphylaxis in mast cell-deficient mice.

Mast cells have long been believed to be the central effector cells in the development of immunoglobulin (Ig)E-dependent anaphylaxis. In this study, we investigated the role of mast cells in IgE-dependent hapten-induced active fatal anaphylaxis using mast cell-deficient WBB6F1- W/Wv (W/Wv) and congenic normal (+/+) mice. Although a 5-min delay in shock signs and death were observed in W/Wv mice, 100% fatal reactions to penicillin V (Pen V) occurred in both +/+ and W/Wv mice. Administration of monoclonal anti-IL-4 antibody completely prevented the fatal reactions, and the effect of anti-IL-4 was associated with its suppressive activity on Pen V-specific serum levels of IgE, but not IgG. The platelet-activating factor (PAF) antagonist, BN 50739, completely prevented the fatal reactions in both strains of mice. Our kinetic study revealed, in contrast to no elevation of plasma histamine level in W/Wv mice, high levels of PAF in the circulation after challenge in both +/+ and W/Wv mice, albeit to a lesser degree in the latter case. These data indicate that cells other than mast cells are sufficient to induce an IgE-dependent active fatal anaphylaxis by elaborating PAF, which is the critical mediator for fatal murine anaphylaxis.

Steroid receptor coactivator-1 interacts with the p50 subunit and coactivates nuclear factor kappaB-mediated transactivations.

Steroid receptor coactivator-1 (SRC-1) specifically bound to the transcription factor NFkappaB subunit p50 but not to p65 as demonstrated by the yeast two hybrid tests and glutathione S-transferase pull down assays. The p50-binding site was localized to a subregion of SRC-1 (amino acids 759-1141) that encompasses the previously described CBP-p300-binding domain. In mammalian cells, SRC-1 potentiated the NFkappaB-mediated transactivations in a dose-dependent manner. Coexpression of p300 further enhanced this SRC-1-potentiated level of transactivations, consistent with the recent findings in which CBP and p300 were shown to be transcription coactivators of the p65 subunit (Perkins, N. D., Felzien, L. K., Betts, J. C., Leung, K., Beach, D. H., and Nabel, G. J. (1997) Science 275, 523-527; Gerritsen, M. E., Williams, A. J., Neish, A. S. , Moore, S., Shi, Y., and Collins, T. (1997) Proc. Acad. Natl. Sci. U. S. A. 94, 2927-2932). These results suggest that at least two distinct coactivator molecules may cooperate to regulate the NFkappaB-dependent transactivations in vivo and SRC-1, originally identified as a coactivator for the nuclear receptors, may constitute a more widely used coactivation complex.

Steroid receptor coactivator-1 coactivates activating protein-1-mediated transactivations through interaction with the c-Jun and c-Fos subunits.

Steroid receptor coactivator-1 (SRC-1) specifically bound to the transcription factor AP-1 subunits c-Jun and c-Fos, as demonstrated by the yeast two-hybrid tests and glutathione S-transferase pull down assays. The c-Jun and c-Fos binding sites were localized to the C-terminal subregion of SRC-1 (amino acids 1101-1441) that encompasses the previously described histone acetyltransferase and receptor-binding domains. In mammalian cells, SRC-1, similar to the previous results with CBP-p300 (Arias, J., Alberts, A. S., Brindle, P., Claret, F. X., Smeal, T., Karin, M., Feramisco, J., and Montminy, M. (1994) Nature 370, 226-229; Bannister, A. J., and Kouzarides, T. (1995) EMBO J. 14, 4758-4762), potentiated the AP-1-mediated transactivations in a dose-dependent manner and derepressed the mutual inhibitions between nuclear receptors and AP-1. Furthermore, coexpression of p300 further enhanced this SRC-1-potentiated level of transactivations. Thus, we concluded that at least two distinct coactivator molecules may cooperate to regulate AP-1-dependent transactivations and mediate transrepression between AP-1 and nuclear receptors in vivo.

Involvement of nuclear factor-kappa B in platelet-activating factor-mediated tumor necrosis factor-alpha expression.

Tumor necrosis factor (TNF)-alpha and platelet-activating factor (PAF) are important mediators of inflammatory reactions, and their release is controlled by a positive feedback network. However, the regulatory mechanisms underlying the interaction of these two molecules are unknown. Within 10 min of the injection of lipopolysaccharide (LPS) into C57BL/6 mice, effects inducible by PAF such as anaphylactic shock-like symptoms, disseminated intravascular coagulation, and hemorrhage in renal medullae were observed, and all these pathological changes were prevented by the PAF antagonist, BN 50739. The plasma level of PAF after LPS injection reached a peak at 5 min. TNF-alpha gene expression was evident 20 min after LPS injection and was maximal at 40 min, and the level of serum TNF-alpha reached a peak at 1 h. Pretreatment with BN 50739 inhibited LPS-induced TNF-alpha gene expression and protein synthesis in a dose-dependent manner. Injection of PAF or treatment of the macrophage cell line, J774A.1, with PAF activated the transcription factor, nuclear factor (NF)-kappa B, which is essential for inducible TNF-alpha transcription. The activation of NF-kappa B by PAF preceded the LPS-mediated TNF-alpha gene expression. Pretreatment with BN 50739 inhibited LPS-induced mobilization of NF-kappa B in a dose-dependent manner in vivo as well as in vitro. These data suggest that PAF, which is released immediately or shortly after LPS injection, induces the expression of TNF-alpha through the activation of NF-kappa B.

Constitutive activation of NF-kappa B in an animal model of aging.

The pathophysiology of many disease states observed in aged individuals has been linked to the dysregulated production of several pleiotropic cytokines. We have demonstrated that NF-kappa B, a major transcriptional regulator of these aberrantly expressed cytokines, exists in a constitutively activated state in cells obtained from the major lymphoid organs of aged animals. Therapeutic treatment with dietary antioxidants or with agents capable of activating the peroxisome proliferator-activated receptor (PPAR)-alpha was able to correct the abnormal nuclear NF-kappa B activity, reduce lipid peroxide levels, and eliminate the dysregulated expression of cytokines and other genes under NF-kappa B control. These results suggest that abnormal activation of NF-kappa B in aging contributes to the dysregulated expression of certain pleiotropic cytokines. Effective therapeutic regimens for aging and other inflammatory disease states might benefit from the administration of antioxidants or other agents which specifically activate PPAR-alpha.

A protective role of platelet-activating factor in murine candidiasis.

Platelet-activating factor (PAF) is a potent phospholipid-derived modulator of immunological and inflammatory processes. In this study, the role of exogenous and endogenous PAF in resistance to infection with Candida albicans was investigated. Administration of PAF following a lethal challenge of C. albicans significantly protected mice from death and reduced the number of organisms in the kidneys. Neutralization of endogenous PAF with the PAF antagonist BN50739 shortened the mean survival time and increased the number of C. albicans cells per kidney. Shortly after infection of mice (30 min), significant levels of PAF were detected in the serum. PAF-induced protection appears to be mediated through the actions of tumor necrosis factor alpha (TNF-alpha), since pretreatment with anti-TNF-alpha before each injection of PAF abrogated the majority of PAF-induced enhanced resistance. Administration of PAF in vivo elevated serum TNF-alpha levels and TNF-alpha mRNA expression in the kidney. Production of TNF-alpha was markedly diminished by pretreatment with the PAF antagonist BN50739 prior to infection with C. albicans. We conclude that PAF, which is produced during infection with C. albicans, plays an important role in determining the level of resistance to this infectious microorganism. This effect of PAF appears to be mediated, at least in part, through the induction of TNF-alpha.

Augmentation of tumor metastasis by platelet-activating factor.

The effect of platelet-activating factor (PAF) on experimental pulmonary metastasis by the B16F10 murine melanoma and the possible involvement of PAF in the activities of tumor necrosis factor alpha (TNF-alpha) and interleukin 1alpha (IL-1alpha) in tumor metastasis were investigated. i.p. injection of PAF enhanced the lung colonization in a dose- and time-dependent manner. PAF enhanced lung colonization when it was administered after, but not before, B16F10 inoculation. Multiple injections of PAF were more effective than a single injection. Neutralization of endogenous PAF with PAF antagonist BN50739 decreased lung colonization, suggesting that endogenous PAF plays an important role in pulmonary metastases. A single i.p. injection of TNF-alpha or IL-1alpha caused a marked enhancement in lung colonization. TNF-alpha- and IL-1alpha-mediated enhancement in lung colonies was significantly inhibited by BN50739. These results demonstrate that PAF has a metastasis-enhancing effect and is a mediator of the metastatic activities of TNF-alpha and IL-1alpha.

Dual effects of pertussis toxin on murine neutrophils in vivo. I. Pertussis toxin inhibits extravasation potential of mature neutrophils while simultaneously stimulating granulopoiesis.

Pertussis toxin (Ptx) has been employed as an adjuvant by many investigators to augment various types of cell-mediated and humoral immune responses. Recent work from our laboratory indicates that the exacerbation of delayed-type hypersensitivity (DTH) and contact hypersensitivity (CH) responses observed in Ptx-treated mice may be mediated by an absolute increase in the number of circulating neutrophils capable of migrating into tissue sites of antigen challenge. The purpose of the present study was to analyze the effects of Ptx on neutrophils and neutrophil function in vivo. Evidence is presented here suggesting that Ptx has both direct and indirect effects on neutrophils following its in vivo administration to normal mice. Mature neutrophils that are directly exposed to the actions of Ptx in vivo exhibit a marked reduction in their ability to extravasate into tissue sites of inflammation. These findings are consistent with those that have been reported following the exposure of isolated neutrophils to the effects of Ptx in vitro (i.e., that Ptx has an inhibitory effect on many of the functional capabilities of isolated neutrophils). Moreover, we have also determined that Ptx can affect the kinetics of neutrophil production indirectly through its ability to stimulate granulopoiesis. Ptx-exposed mice develop a protracted peripheral blood neutrophilia following toxin administration. Although the mechanism(s) involved in stimulating increased neutrophil production is presently unclear, both dexamethasone and indomethacin (cyclooxygenase pathway inhibitors) are able to function synergistically with Ptx to produce a markedly enhanced neutrophilia in exposed mice. We propose that the capacity of Ptx to augment CH and DTH responses and act as a potent adjuvant may relate, in part, to its ability to alter the rate of neutrophil production in vivo.

Molecular mechanisms of lymphocyte extravasation. III. The loss of lymphocyte extravasation potential induced by pertussis toxin is not mediated via the activation of protein kinase C.

The present study evaluated whether protein kinase C (PKC) activation was involved in the lymphocytosis promoting properties of pertussis toxin (Ptx). The exposure of mouse lymphocytes to phorbol esters (as a means to selectively activate PKC) caused a depression in their subsequent capacity to localize into lymph nodes and Peyer's patches in vivo. This pattern of inhibition was quite similar to that observed with lymphocytes treated with Ptx. The mechanisms responsible for the observed decreases in localization to lymphoid organs caused by these two agents, however, appeared to be distinct. Exposure of lymphocytes to PMA was followed by a time and dosage-dependent decrease in the surface density of MEL-14 defined homing receptors. Ptx-treated lymphocytes retained normal density of this homing receptor. Consequently, PMA-treated lymphocytes lost their capacity to bind to high-endothelial venules in in vitro lymph node binding assays while Ptx-treated cells retained normal high-endothelial venule binding potential. We conclude from this study that: 1) the activation of PKC in lymphocytes by PMA can alter their recirculation properties via mechanisms that diminish their expression of surface receptors which support extravasation into lymph node and mucosal lymphoid tissues, and 2) even though Ptx has been reported to elevate the rate of inositol phosphate turnover in lymphocytes, the loss of extravasation potential of Ptx-treated lymphocytes is not mediated via the modification of surface homing receptors as observed in cells exposed to the known PKC activator, PMA.