NKRP1A molecule is involved in transendothelial migration of CD4+ human T lymphocytes

Among human CD4+ T lymphocytes, 5-20% express the C-type lectin molecule NKRP1A. Interestingly, CD4+ NKRP1A+ T lymphocytes express high levels of beta 1 and beta 2 integrins, thus representing a T lymphocyte subset that can possibly adhere and migrate through vascular endothelium. Indeed, resting CD4+ NKRP1A+ lymphocytes, differently from the CD4+ NKRP1A- subset, migrated across endothelial cell monolayers in a Transwell chamber system. This transendothelial migration was strongly reduced after pre-treatment with an anti-NKRP1A monoclonal antibody (mAb). In addition, the NKRP1A negative Jurkatt CD4+ T-cell line that had been stably transfected with NKRP1A cDNA, migrated more rapidly and efficiently than untransfected Jurkatt cells. Finally, mAb-mediated cross-linking of NKRP1A molecule in CD4+ T lymphocytes induced the upregulation of the LFA1 Mg2+ binding site as well as beta 1 and beta 2 integrin chains. Altogether, these findings indicate that NKRP1A molecule is involved in transendothelial migration of resting CD4+ T lymphocytes.     

TNF-alpha released by comigrating monocytes promotes transendothelial migration of activated lymphocytes

We investigated mechanisms that increase motility and transendothelial trafficking of activated lymphocytes. Freshly isolated lymphocytes stimulated with immobilized anti-CD3 for 2 h migrate into polymerized collagen in 1.99+/-0.25-fold greater numbers and across confluent endothelial monolayers in 4.8+/-0.5-fold greater numbers compared with leukocytes incubated with non-specific IgG. Activated lymphocytes form clusters with monocytes, and their increased motility was dependent on the presence of comigrating monocytes. Five lines of evidence support the idea that monocytes modulate lymphocyte motility through the release of TNF-alpha: 1) flow-cytometric analyses, using highly specific and avid mAbs to probe permeabilized whole blood leukocytes, showed that >80% of circulating monocytes contain intracellular TNF-alpha, whereas <5% contain IL-1 and none contain IL-6; 2) stimulation with immobilized anti-CD3 that was intended to activate lymphocytes also induced monocytes to release increased quantities of TNF-alpha; 3) rTNF-alpha, added in doses of 1 to 20 pg/ml to purified anti-CD3-stimulated lymphocytes, reproduced, in a dose-dependent manner, the motility-enhancing effect of adding monocytes; 4) the transient increase in the expression of TNF R-I on CD3-activated T lymphocytes parallels their transiently increased motility; and 5) addition of anti-TNF-alpha, anti-TNF R-I, anti-TNF R-II, or soluble TNF R-I decreased the motility of stimulated lymphocytes. These results suggest that T lymphocyte stimulation via the CD3-TCR complex signals nearby monocytes to release TNF-alpha, which feeds back on the lymphocytes to increase their locomotor activity.     

Inhibition of prostaglandin E release by anti-inflammatory steroids

The aearance of tritium in plasma water was measured after i.p. injection of 2-tritio-L-alanine as an index of alanine transamination in rats. L-Cycloserine (10 mg/kg) and D-cycloserine (150 mg/kg) inhibited tritium release, whereas theophylline (100 mg/kg) stimulated tritium release.     

Inhibition of voltage-dependent calcium channels by prostaglandin E2 in rat melanotrophs

The effects of PGE2 on voltage-dependent Ca2+ channel currents were studied in dissociated rat melanotrophs by the whole-cell configuration of the patch-clamp technique. In about 90% of melanotrophs examined, PGE2 reversibly inhibited voltage-dependent Ba2+ currents elicited by voltage steps from a holding potential of -80 to 0 mV, with an ED50 of 68 nM. The maximum inhibition of Ba2+ currents by 1 microM PGE2 (35.3%) was comparable with that by the maximally effective concentration (100 nM) of dopamine. The EP1/EP3 PGE (EP) agonists, 17PT-PGE2 and sulprostone, and the EP2/EP3 agonist, misoprostol, mimicked the inhibition by PGE2, whereas the selective EP2 agonist, butaprostol, had little effect. The inhibition by PGE2 was partially, but significantly, reduced by the selective EP1 antagonist, SC-51322. The magnitude of the PGE2-induced inhibition of Ba2+ currents was greatly reduced by pretreatment with pertussis toxin, or by a depolarizing prepulse, to +80 mV, lasting for 50 msec. Although four distinct types (N-, P/Q-, L-, and R-types) of high-threshold Ba2+ currents were observed, PGE2 (1 microM) caused significant inhibition of only P/Q- and L-type currents, which were 17.3 and 10.1%, respectively, of the total Ba2+ currents. These results suggest that PGE2 inhibits P/Q- and L-type Ca2+ channels of rat melanotrophs via EP1 and EP3 receptors, which are coupled to pertussis toxin-sensitive G proteins, and produces both voltage-sensitive and -insensitive inhibition of Ca2+ channels.     

Inhibition of human oral squamous carcinoma cell (SCC-25) proliferation by prostaglandin E2 and vitamin E succinate

The primary objective of this investigation was to study the effect of D-alpha-tocopherol acid succinate (vitamin E succinate) and prostaglandin E2 (PGE2), individually and in combination, on the proliferation of human tongue squamous carcinoma cells (SCC-25) in vitro. Test compounds in varying concentrations were incubated with cells in serum-free Dulbecco's Modified Eagle's Medium-Ham's F-12 Medium (50:50), supplemented with 0.1% albumin for sixteen hours. Cell proliferation was measured by the incorporation of [3H] thymidine in acid-insoluble material (i.e. DNA). Prostaglandin E2 and vitamin E succinate, individually at 10(-9)-10(-6) M, caused significant dose-dependent inhibition in DNA synthesis. A combined dose of each compound at 10(-5) M resulted in significant additive inhibition which averaged 43.53% (p < 0.005). Addition of indomethacin (INDO) to cell cultures induced significant dose-dependent stimulation in DNA synthesis. Hence, we might suggest that the overall potential of vitamin E in controlling malignant cell proliferation in vivo could be due to its own effect combined with that of endogenous PGs which are normally produced in excessive amounts by malignant cells.     

Inhibition of EGF-dependent mitogenesis by prostaglandin E2 in Syrian hamster embryo fibroblasts

Efficient syntheses of 4-(R,S-hydroxyphosphonomethyl)-L-phenylalanine and 4-carboxy-L-phenylalanine within the context of the pentapeptide Ac-Ile-X-Gly-Glu-Phe-NH2 (wherein X = the unnatural amino acid) illustrate the use of a divergent synthetic strategy from an advanced common peptide intermediate to more readily access peptide-based tyrosine kinase inhibitors. The key intermediate, Ac-Ile-Phe(4-formyl)-Gly-Glu(O-tBu)-Phe-NH2, was synthesized by a facile palladium-catalyzed carbonylation of Ac-Ile-Phe(4-iodo)-Gly-Glu(O-tBu)-Phe-NH2. Oxidation of Ac-Ile-Phe(4-formyl)-Gly-Glu(O-tBu)-Phe-NH2 with tetrabutylammonium permanganate or addition of di-t-butylphosphite, both followed by trifluoroacetic acid deprotection, gave the target pentapeptide inhibitors wherein X = 4-carboxy-L-phenylalanine or 4-(R,S-hydroxyphosphonomethyl)-L-phenylalanine, respectively. These two peptides gave somewhat more potent inhibition of the tyrosine kinase pp60c-src than the corresponding pentapeptide wherein X = L-phenylalanine, demonstrating that appended functionalities at the 4-position are accepted and can enhance binding through added interactions within the catalytic region of the active site.     

Antigen recognition influences transendothelial migration of CD4+ T cells

The functional significance of MHC class II expression by vascular endothelial cells remains obscure. In this study the possibility that Ag presentation by endothelial cells (EC) influences T cell transmigration, facilitating the recruitment of Ag-specific T cells into tissues, was investigated. The frequencies of T cells with specificity for an HLA-DR alloantigen, or for the recall Ag tetanus toxoid (TT), were measured in peripheral blood CD45RO+ (memory) CD4+ T cells before and after transmigration through gamma-IFN-treated EC monolayers. Frequencies of anti-DR17, IL-2-secreting T cells were fourfold higher in the T cells that transmigrated through a monolayer of DR17-expressing EC. Similar increases were seen in TT-specific, DR7-restricted T cells that transmigrated through TT-pulsed, DR7-expressing EC. To examine more directly the effects of cognate recognition of Ag presented by EC, T cell clones were used. For clones that proliferated in a costimulation-independent manner to Ag presented by EC, cognate recognition arrested transmigration. In contrast, Ag presentation by EC to B7-dependent T cell clones, which do not proliferate following cognate recognition of EC, enhanced the rate of transendothelial migration. These data suggest that Ag presentation by EC may serve to augment the recruitment of Ag-specific T cells into tissues and that proliferation and transmigration are mutually exclusive T cell responses.     

Reduction of the stretch modulus of human cervical tissue by prostaglandin E2

Samples of cervical tissue were obtained from women immediately following term delivery from spontaneous labor or oral PGE2-induced labor and from a small nunber of nonpregnant women in the reproductive years undergoing hysterectomy for benign gynecologic disease. The measured strips were placed in a well-oxygenated bath at 37 degrees C. containing Ringer's solution and stretched at a constant rate while continuously recording length and tension. The data were converted to stress-strain diagrams. Each curve contained a long linear portion which allowed one to compute a stretch modulus for each sample. A total of 71 strips from 23 patients was used. The average stretch modulus from the oral PGE2-induced patients was significantly lower than the spontaneous labor group (p less than 0.005), which, in turn, was significantly lower than the stretch modulus of the nonpregnant cervical tissue. It was also noted that the yield point was lower in the PGE2 series when compared to the spontaneous labor series (p less than 0.05). The effect of the PGE2 at a bath concentration of 10(-5) to 10(-6)Gm. per cubic centimeter was to materially reduce the stretch modulus within 5 to 15 minutes of the drug addition in both the PGE2-induced and spontaneous labor series. The results of these experiments indicate that PGE2 has the effect of reducing cervial stiffness.     

Inhibition of sodium transport by prostaglandin E2 across the isolated, perfused rabbit collecting tubule

This study was designed to examine whether prostaglandin E2 can directly affect sodium transport across isolated perfused rabbit renal collecting tubules. Changes in transepithelial potential and isotopic sodium fluxes in response to peritubular prostaglandin E2 were measured. In addition, changes in transepithelial potential of the outer medullary collecting tubule in response to prostaglandin E2 were also measured. With few exceptions, all rabbits received 5 mg/day desoxycorticosterone acetate for 4-11 days before experimentation. The results of the experiments show that: (a) prostaglandin E2 inhibits the negative transepithelial potential in the cortical collecting tubule as well as the outer medullary collecting tubule; (b) prostaglandin E2 inhibits net sodium transport out of the lumen by inhibiting efflux while backflux is unaffected; (c) prostaglandin E2 produces this inhibition within 15 min, and the effects are dose dependent and reversible. These results suggest that prostaglandin E2 may modulate sodium transport in vivo and may contribute to the final regulation of sodium excretion.     

Activated T cells acquire endothelial cell surface determinants during transendothelial migration

Activated T cells acquire endothelial cell (EC) plasma membrane constituents during transendothelial migration. This was assessed using an in vitro model system in which human peripheral blood CD4+ T cells migrated through confluent monolayers of HUVEC. Flow cytometry of migrated CD4+ T cells demonstrated that activated, but not resting, T cells acquired a variety of endothelial surface determinants, including CD31, CD49d, CD54, CD61, and CD62E. The extracellular domains of these molecules were detected on migrated T cells with mAbs, including those directed to the ligand-binding regions. A number of approaches were employed to document that the acquisition of these molecules was uniquely accomplished by activated T cells and clearly involved transfer from both resting and TNF-alpha-activated EC. Acquisition of endothelial markers by activated T cells occurred as part of the transfer of membrane components, as migrating T cells acquired EC membranes prelabeled with the lipophilic dye, 3,3'-dihexadecyloxacarbocyanine perchlorate (DiOC-16), along with EC surface proteins. Thus, during transendothelial migration, activated T cells acquire endothelial membrane components, and as a result may deliver them to perivascular sites.     

Inhibition of cyclooxygenase-2 rapidly reverses inflammatory hyperalgesia and prostaglandin E2 production

PGs derived from cyclooxygenase-2 (COX-2), in particular PGE2, play important roles in the initiation of inflammation and pain. In the present study, we evaluated the role of COX-2-derived PGE2 in an animal model of established hyperalgesia. Inflammation and hyperalgesia were first induced by injection of carrageenan into rat footpads. Then we investigated the effects of subsequent therapeutic treatment with a selective COX inhibitor, with a nonsteroidal anti-inflammatory drug and with anti-PGE2 antibody. Test compounds were administered 1 to 3 hr after carrageenan challenge, and inhibition of pain (hyperalgesia, measured by withdrawal from a thermal stimulus), and changes in paw edema and PG levels were evaluated. The i.v. administration of a nonselective COX inhibitor, ketorolac, caused a rapid reduction in hyperalgesia in the inflamed footpad, returning it to near-normal values within 1 hr. Normal (control) paw response times were not affected. Therapeutic administration of ketorolac prevented most further swelling caused by carrageenan but did not reverse edema already present at the time of dosing. Administered p.o., a selective COX-2 inhibitor (SC-58635) was as efficacious as ketorolac in reducing inflammatory hyperalgesia. Footpad PG levels returned to base line or below within 5 min of dosing with ketorolac, which suggests rapid turnover of PG in the inflamed tissue. Therapeutic treatment with a monoclonal anti-PGE2 antibody also fully reversed the hyperalgesia response. These studies suggest that continuous production of PGE2 by the COX-2 enzyme is a critical element in sustaining the hyperalgesic response at sites of tissue inflammation.     

Inhibition of purinergic transmission by prostaglandin E1 and E2 in the guinea-pig vas deferens: an electrophysiological study

1. The effects of prostaglandin E1 (PGE1) and E2 (PGE2) on postjunctional electrical activity in the guinea-pig vas deferens evoked by sympathetic nerve stimulation were investigated using both intracellular and focal extracellular recording techniques in vitro. 2. Bath application of PGE1 (1-100 nM) or PGE2 (0.1-100 nM) concentration-dependently inhibited the amplitudes of all excitatory junction potentials (e.j.ps) evoked during short trains of stimuli (10 stimuli at 1 Hz). Increasing the duration of nerve stimulation (100 stimuli at 1 Hz) did not overcome this inhibitory effect. At these concentrations PGE1 and PGE2 were without any apparent inhibitory effect on the amplitudes of spontaneous e.j.ps. 3. Local application of PGE1 (10-100 nM) or PGE2 (10-30 nM) markedly reduced the frequency of occurrence of excitatory junction currents (e.j.cs) evoked by trains of 20-100 stimuli at 1 to 4 Hz without changing the amplitudes of spontaneous e.j.cs or the configuration of the nerve terminal impulse. 4. In the presence of PGE1 or PGE2, raising the frequency of stimulation (from 1 to 4 Hz), increased the likelihood of e.j.c. occurrence. 5. The postjunctional electrical activity recorded in the guinea-pig vas deferens is believed to be due to ATP released from the sympathetic nerve endings. Thus the present study demonstrates that both PGE1 and PGE2 powerfully inhibit quantal ATP release in the guinea-pig vas deferens.     

Induction of cyclo-oxygenase-2 mRNA by prostaglandin E2 in human prostatic carcinoma cells

Prostaglandins are synthesized from arachidonic acid by the enzyme cyclo-oxygenase. There are two isoforms of cyclooxygenases: COX-1 (a constitutive form) and COX-2 (an inducible form). COX-2 has recently been categorized as an immediate-early gene and is associated with cellular growth and differentiation. The purpose of this study was to investigate the effects of exogenous dimethylprostaglandin E2 (dmPGE2) on prostate cancer cell growth. Results of these experiments demonstrate that administration of dmPGE2 to growing PC-3 cells significantly increased cellular proliferation (as measured by the cell number), total DNA content and endogenous PGE2 concentration. DmPGE2 also increased the steady-state mRNA levels of its own inducible synthesizing enzyme, COX-2, as well as cellular growth to levels similar to those seen with fetal calf serum and phorbol ester. The same results were observed in other human cancer cell types, such as the androgen-dependent LNCaP cells, breast cancer MDA-MB-134 cells and human colorectal carcinoma DiFi cells. In PC-3 cells, the dmPGE2 regulation of the COX-2 mRNA levels was both time dependent, with maximum stimulation seen 2 h after addition, and dose dependent on dmPGE2 concentration, with maximum stimulation seen at 5 microg ml(-1). The non-steroidal anti-inflammatory drug flurbiprofen (5 microM), in the presence of exogenous dmPGE2, inhibited the up-regulation of COX-2 mRNA and PC-3 cell growth. Taken together, these data suggest that PGE2 has a specific role in the maintenance of human cancer cell growth and that the activation of COX-2 expression depends primarily upon newly synthesized PGE2, perhaps resulting from changes in local cellular PGE2 concentrations.     

Modulators of prostaglandin E2 synthesis in human amnion

OBJECTIVE: The purpose of these studies was to determine the effects of the essential fatty acid, linoleic acid, and the commonly used non-steroidal anti-inflammatory agents, aspirin and acetaminophen, on the rate of prostaglandin (PG) biosynthesis by human amnion cells. METHODS: Amnion cells were isolated from term, normal pregnancies and grown to confluence. Cells were incubated with control or medium containing 100 mumol/L linoleic acid. Cells were also incubated with control medium or medium containing 10 or 100 micrograms/mL aspirin or acetaminophen. RESULTS: Following an initial delay, amnion cells exposed to linoleic acid exhibited a significant increase in PGE synthesis. Both aspirin and acetaminophen in clinically relevant concentrations had a significant inhibitory effect on amnion cell PGE synthesis. CONCLUSIONS: Linoleic acid has a stimulatory effect and aspirin and acetaminophen have an inhibitory effect on PGE synthesis in human amnion cells in culture. We speculate that dietary habits, supplement ingestion, and over-the-counter drug use may affect amnion cell PG production. In view of the potential importance of intrauterine PG production in normal and abnormal labor, further study in this area is indicated.     

Phenotypic and functional analysis of CD4+ NKRP1A+ human T lymphocytes. Direct evidence that the NKRP1A molecule is involved in transendothelial migration

In this report, we show that among human CD4+ T lymphocytes 5-20% express the C-type lectin molecule NKRP1A. This lymphocyte subset displays a slightly more limited T cell receptor V beta repertoire than the CD4+ NKRP1A- counterpart. CD4+ NKRP1A+ T lymphocytes are characterized by a high expression of beta 1 and beta 2 integrins, thus representing a T lymphocyte subset that can possibly adhere and migrate through vascular endothelium. Indeed, resting CD4+ NKRP1A+ lymphocytes, differently from the CD4+ NKRP1A- subset, migrated across endothelial cell monolayers in a Transwell chamber system. Pretreatment of CD4+ NKRP1A+ T lymphocytes with an anti-NKRP1A monoclonal antibody (mAb) strongly reduced transendothelial migration, suggesting the involvement of the NKRP1A molecule in the transmigration process. Furthermore, cells of the NKRP1A- Jurkat CD4+ T cell line stably transfected with NKRP1A cDNA migrated more rapidly and efficiently than either untransfected or mock-transfected Jurkat cells. Finally, mAb-mediated cross-linking of NKRP1A molecules in CD4+ T lymphocytes induced the up-regulation of the lymphocyte function-associated antigen 1 Mg(2+)-binding site as well as beta 1 and beta 2 integrin chains. Altogether, these findings suggest that the NKRP1A molecule is involved in transendothelial migration of resting CD4+ T lymphocytes.     

Induction by interleukin-1beta peptide of prostaglandin E2 formation via enhanced prostaglandin H synthase-2 expression in 3T6 fibroblasts

Several synthetic interleukin-1 (IL-1) peptides were tested in vivo for pyrogenic activity and in vivo for their ability to stimulate prostaglandin production. Only the IL-1beta fragment (208-240) enhanced body temperature, although both IL-1beta (208-240) and IL-1alpha (223-250) stimulated prostaglandin E2 (PGE2) production in vitro. We report here that the IL-1beta fragment (208-240) did not have the capacity to induce arachidonic acid (AA) mobilization by 3T6 fibroblasts. However, this peptide was able to increase the expression of the inducible prostaglandin H synthase isoform (PGHS-2; EC 1.14.99.1.), which is related to its ability to stimulate prostaglandin E2 synthesis.     

Cell shape changes and cytoskeleton reorganization during transendothelial migration of human melanoma cells

An in vitro system has been established to study the migration of human melanoma cells through a monolayer of endothelial cells. Endothelial cells were cultured to confluence on Matrigel before the seeding of melanoma cells. Laser scanning confocal microscopy showed that, prior to migration, melanoma cells appeared round and showed cortical F-actin staining. The initial stage of transmigration was characterized by numerous membrane blebs protruding from basolateral surfaces of the melanoma cells, and contact regions showed an abundance of filaments arising in the underlying endothelial cells. Later, pseudopods from the melanoma cells inserted into contact regions between endothelial cells. Eventually, the melanoma cells intercalated with the endothelial cells. At this stage, many endothelial filament bundles terminated at contacts between the endothelial cells and the transmigrating melanoma cell, suggesting active interactions between the two cell types. Upon contact with the Matrigel, melanoma cells began to spread beneath the endothelium, displaying a fibroblastic morphology with prominent stress fibers. To reestablish the monolayer, adjacent endothelial cells extended processes over the melanoma cell. Tumor necrosis factor alpha did not affect the transmigration of melanoma cells from cell lines isolated from several stages of metastasis. However, tumor necrosis factor did promote the transmigration of melanoma cells derived from a non-metastatic lesion. These results thus define cell attachment and cell penetration of the monolayer as two distinct steps in transmigration and suggest that tumor necrosis factor may enhance the metastatic potential of tumor cells.     

Prostaglandin E2 in human placenta: its vascular effects and activation of prostaglandin E2 formation by nicotine and cotinine

Tobacco smoking by pregnant women increases the frequency of spontaneous abortions and preterm births. Human labor is associated with enhanced intrauterine phospholipid metabolism and production of prostaglandin E2 (PGE2) which induces labor, initiates uterine contractions and maintains the homeostasis of placental blood flow. Therefore, we studied: (a) the influence of nicotine and cotinine on the effects of PGE2 on placental vasculature in perfused human placental cotyledon, and (b) the activation of placental phospholipase A2 (PLA2) by nicotine and cotinine using 1-palmitoyl-2-[1-14C]arachidonyl-phosphatidylethanolamine (PE, 2.2 nmol) as substrate. These studies revealed that: (1) increasing concentrations of PGE2 (10- 150 ng/ml) increased umbilical perfusion pressure by 170 +/- 10% (n = 6) of control (100%). Cotinine (2 microg/ml) enhanced this effect at all concentrations of PGE2. Nicotine (2 microg/ml) prevented the effect of PGE2; (2) both cotinine (EC50 470-500 fmol/l) and nicotine (EC50 18-32 pmol/l) activated PLA2 in human placental tissues. These observations indicated that cotinine was more potent than in nicotine activating PLA2 and potentiating the vasoconstrictive effects of PGE2 on fetal placental circulation. Nicotine activates nicotinic receptors and releases placental acetylcholine, a vasodilator of placental arteries. Acetylcholine stimulates muscarinic receptors of endothelial cells resulting in the release of endothelium-derived relaxing factor (EDRF), and possibly nitric oxide. Therefore, nicotine prevents or abolishes the vasoconstrictive effects of PGE2 through the release of EDRF. Cotinine is inactive at nicotinic and muscarinic receptors. Therefore, accumulation of cotinine, the major metabolite of nicotine, in fetal circulation may contribute to production of PGE2 and induction of preterm labor and spontaneous abortions.