An IFNG SNP with an estrogen-like response element selectively enhances promoter expression in peripheral but not lamina propria T cells.

This study examines mucosa-specific regulatory pathways involved in modulation of interferon-gamma (IFN-gamma) in lamina propria T cells. Previous studies identified mucosa-specific CD2 cis-elements within the -204 to -108 bp IFNG promoter. Within this region, a single-site nucleotide polymorphism, -179G/T, imparts tumor necrosis factor-alpha stimulation of IFNG in peripheral blood lymphocytes, and is linked with accelerated AIDS progression. We discovered a putative estrogen response element (ERE) introduced by the -179T, which displays selective activation in peripheral blood mononuclear cells (PBMC) vs lamina propria mononuclear cells (LPMC). Transfection of PBMC with constructs containing the -179G or -179T site revealed CD2-mediated enhancement of the -179T compared to -179G allele, although, in LPMC, a similar level of expression was detected. Electrophoretic mobility shift assay (EMSA) analysis demonstrated CD2-mediated nucleoprotein binding to the -179T but not the -179G in PBMC. In LPMC, binding is constitutive to both -179G and -179T regions. Sequence and EMSA analysis suggests that the -179T allele creates an ERE-like binding site capable of binding recombinant estrogen receptor. Estrogen response element transactivation is enhanced by CD2 signaling, but inhibited by estrogen in PBMC but not in LPMC, although expression of estrogen receptor was similar. This is the first report to describe a potential molecular mechanism responsible for selectively controlling IFN-gamma production in LPMC.

Do children from an inner city British school meet the recommended levels of physical activity? Results from a cross sectional survey using objective measurements of physical activity.

Questionnaire surveys suggest physical activity levels in children are low, particularly among children from deprived areas. Using accelerometers, it was found that children from a deprived inner city school were active at recommended levels and had similar levels of activity to children in other studies from more affluent populations. However, this finding was dependent on the threshold used to define moderate activity.

CCR9-positive lymphocytes and thymus-expressed chemokine distinguish small bowel from colonic Crohn's disease.

BACKGROUND & AIMS: Thymus-expressed chemokine (TECK) or CCL25) is selectively expressed in the small bowel (SB), where lamina propria lymphocytes (LPL) and intraepithelial leukocyte expressing the cognate chemokine receptor CCR9 predominate. We characterize the role of TECK and CCR9-expresing lymphocytes in small intestinal Crohn's disease. METHODS: CCR9 expression on lymphocytes from lamina propria, mesenteric lymph node, and peripheral blood was analyzed by flow cytometry and by Northern blotting for LPL. TECK expression was analyzed in inflamed SB and colon by reverse-transcription polymerase chain reaction and immunohistochemistry. RESULTS: The fraction of CCR9(+) T cells in inflamed SB was significantly lower than in uninvolved SB mucosa. In contrast, in peripheral blood lymphocytes, CCR9(+) lymphocytes were markedly elevated in patients with small bowel Crohn's or celiac disease, but not in patients with purely colonic Crohn's. Also, TECK expression is altered in inflamed small bowel, being intensely expressed in a patchy distribution in crypt epithelial cells in proximity to lymphocytic infiltrates. TECK is not expressed in either normal or inflamed colon. CONCLUSIONS: In SB immune-mediated diseases, there is repartitioning of CCR9(+) lymphocytes between SB and blood and an altered pattern of TECK expression in SB Crohn's. The TECK/CCR9 ligand/receptor pair may play an important role in the pathogenesis of SB Crohn's disease.

Mucosa-specific targets for regulation of IFN-gamma expression: lamina propria T cells use different cis-elements than peripheral blood T cells to regulate transactivation of IFN-gamma expression.

Activation of lamina propria (LP) T cells via the CD2 pathway enhances IFN-gamma (IFN-gamma) secretion with further enhancement after CD28 coligation. The molecular mechanisms regulating IFN-gamma expression in LP T cells remain unknown. Previous studies in PBL and T cell lines identified cis- and trans-regulatory elements in TCR-mediated expression of IFN-gamma. This study examines CD2 and PMA/ionophore-responsive IFN-gamma promoter elements. Activation of LPMC via CD2-induced IFN-gamma secretion and a parallel up-regulation of mRNA expression. CD28 coligation enhanced mRNA stability without up-regulating transcription as measured by nuclear run-on. Transfection of a -2.7-kb IFN-gamma promoter-reporter construct into PBL and LP mononuclear cells (LPMC) revealed significant promoter activity after CD2 activation, with additional transactivation after CD2/CD28 costimulation in PBL, but not in LPMC. Functional analysis using truncated promoter fragments identified distinct cis-regulatory regions selectively transactivating IFN-gamma expression in PBL compared with LPMC. In PBL, CD2 activation elements reside within the -108- to +64-bp region. However, in LPMC the upstream region between -204 and -108 bp was essential. Transfection of the proximal and distal AP-1-binding elements, as well as TRE/AP-1 constructs, revealed functional activation of AP-1 subsequent to CD2 signaling, with activation critical in PBL but diminished in LPMC. Electromobility shift analysis using oligonucleotides encompassing the proximal, distal, and BED/AP-1-binding regions failed to demonstrate selective transactivation after CD2 signaling of LPMC. This report provides evidence that activation of LPMC results in transactivation of multiple promoter elements regulating IFN-gamma expression distinct from those in PBL.

CD28 costimulation augments IL-2 secretion of activated lamina propria T cells by increasing mRNA stability without enhancing IL-2 gene transactivation.

The pathways leading to activation in lamina propria (LP) T cells are different from peripheral T cells. LP T cells exhibit enhanced IL-2 secretion when activated through the CD2 pathway. Coligation of CD28 leads to synergistic enhancement of IL-2 secretion. Previous studies have characterized the CD28 augmentation of TCR-mediated signaling in peripheral blood T cells through transcriptional activation of an IL-2 promoter CD28 response element (CD28RE), along with enhanced mRNA stability. This study characterized molecular events involved in CD28 costimulation of IL-2 production in LP mononuclear cells (LPMC). LPMC exhibited increased IL-2 production in response to CD28 costimulation, compared with cells activated through CD2 alone. IL-2 secretion was paralleled by increased expression of IL-2 mRNA, resulting from enhanced IL-2 mRNA stability. In contrast to transcriptional activation in PBMC, EMSA revealed that CD28 coligation of CD2-activated LPMC does not result in increased binding of trans-factors to the CD28RE, nor did Western blots detect changes in I-kappaBalpha or I-kappaBbeta levels following CD28 coligation. Furthermore, CD28 coligation fails to enhance IL-2 promoter-reporter or RE/AP construct expression in CD2-activated LPMC. The results reported herein indicate that the molecular mechanisms involved in CD28 cosignaling and regulation of IL-2 secretion in LP T cells are unique to that compartment and differ from those seen in peripheral blood T cells. These observations suggest a biological significance for different mechanisms of IL-2 activation in initiation and maintenance of the cytokine repertoire found in the mucosa.

Activation of the CD2 pathway in lamina propria T cells up-regulates functionally active AP-1 binding to the IL-2 promoter, resulting in messenger RNA transcription and IL-2 secretion.

The aim of this study was to identify molecular mechanisms involved in transcriptional regulation of IL-2 expression following CD2 and CD3 activation in lamina propria (LP) T cells. Studies used T cells from normal, ulcerative colitis, and Crohn's disease mucosa and freshly isolated PBMC, PBMC stimulated with IL-2 alone, and PBMC stimulated with IL-2 and cocultured with B cell lines (LP-like T cells). Electrophoretic mobility shift assays were performed with nuclear extracts from cells activated with either anti-CD2 or anti-CD3 Abs. CD2 signaling in LPMC and LP-like T cells led to a pattern of sustained up-regulation of AP-1-binding complexes, whereas CD3 activation resulted in only transient up-regulation. While the pattern of regulation of AP-1 binding observed in normal, uninflamed, or inflamed Crohn's disease LPMC is similar, differences in intensity of AP-1 binding were observed. Activation of LP-like T cells mimics the up-regulation of AP-1 with a kinetic profile similar to that observed with freshly isolated LPMC from Crohn's disease-inflamed tissue. The AP-1 complex formed following CD2 activation is composed of jun/fos heterodimers. The CD2-enhanced responsiveness is reflected in functional analysis experiments utilizing transfection of both multimeric-TRE or IL-2 promoter-luciferase constructs directly into normal, ulcerative colitis, or Crohn's disease LPMC. Our data suggest that activation of LP T cells from normal, ulcerative colitis, or Crohn's disease mucosa through the CD2 pathway leads to induction of AP-1 complexes that bind to the IL-2 promoter, and may play a pivotal role in modulating IL-2 production in the gut.

Potent tumoricidal effects of a human cytotoxic T-cell line (TALL-104) against prostate cancer.

The human TALL-104 cell line possesses major histocompatibility complex non-restricted cytotoxic activity against a large variety of tumor targets. Adequate therapies for prostate cancer that has spread outside its capsule are lacking. In order to identify effective therapies for this problem, we investigated the antiproliferative effects of TALL-104 cells against three prostate cancer cell lines (LNCaP, PC-3, DU-145). A Cr-51-release: cytotoxicity assay showed that TALL-104 cells were very cytotoxic against the prostate cancer cells. For example, at a 1:1 ratio of TALL-104 cells to prostate cancer cells, the percent release of Cr-51 at 18 h were 50, 40, and 45% for LNCaP, PC-3, and DU-145, respectively. Analysis by inhibition of clonogenic growth of prostate cancer cells also showed that TALL-104 cells were extremely effective. For instance, a short-term (4 h or 18 h) pre-incubation of TALL-104 cells with these tumor cells at the effector to target ratio of 10:1 prior to clonogenic assay resulted in a substantial reduction in clonogenic tumor growth (90%, 65%, and 50% clonal growth inhibition for LNCaP, PC-3, and DU-145, respectively). Further experiments using both Cr-51 release and clonogenic assays showed that irradiated TALL-104 cells were also effective in their anti-prostatic cancer activities. We also examined if TALL-104 cells plus a chemotherapeutic agent might complement each other in their cytotoxic effects. Preincubation of prostate cancer cell targets with etoposide (0.2-20 mu g/ml) for 18 h markedly increased their susceptibility to TALL-104 lysis. The anti-tumor efficacy of TALL-104 cells was also demonstrated in vivo utilizing the BNX murine model engrafted with subcutaneous PC-3 prostate cancer cells. A substantial reduction in PC-3 tumor cell progression was observed in mice injected with irradiated TALL-104 cells (1x10(7) cells intraperitoneally or intratumorally for 5 days beginning on days 24 and 45 after implantation) as compared to mice injected with tumors only. Taken together, these findings suggest that TALL-104 cells may be utilized as a potent anti-tumor agent, either alone or in combination with other agents (such as etoposide) in metastatic prostate cancer.

A role for TNF-alpha and mucosal T helper-1 cytokines in the pathogenesis of Crohn's disease.

Recent clinical studies of Crohn's disease patients demonstrated dramatic clinical responses following one i.v. infusion of a chimeric mAb to TNF-alpha (cA2). To assess the role of TNF-alpha in mucosal cytokine regulation, the effects of TNF-alpha on lamina propria mononuclear cell (LPMC) Th1 production were determined. Increased IFN-gamma production was demonstrated in anti-CD2-stimulated LPMC cultured in TNF-alpha. To determine the effects of cA2 on cytokine production, TNF-alpha- and IFN-gamma-producing cells were quantitated in LPMC from five Crohn's disease patients treated with cA2. In all four patients who demonstrated clinical and endoscopic improvement, decreased numbers of LPMC producing IFN-gamma and TNF-alpha following CD2/CD28 activation paralleled improvement in disease activity over 8 wk. In one patient who did not improve, increased numbers of TNF-alpha- and IFN-gamma-secreting LPMC were observed. In three of four responding patients, CD2/CD28-activated PBMC demonstrated increased IFN-gamma production over 8 wk. These observations suggest that TNF-alpha may be a cofactor for mucosal Th1 responses, and improvement in clinical parameters and intestinal inflammation induced by cA2 in Crohn's disease may be mediated by down-regulation of mucosal Th1 cytokines.

The expression of tumor necrosis factor in human adipose tissue. Regulation by obesity, weight loss, and relationship to lipoprotein lipase.

A previous study reported the increased expression of the cytokine TNF in the adipose tissue of genetically obese rodents. To examine this paradigm in humans, we studied TNF expression in lean, obese, and reduced-obese human subjects. TNF mRNA was demonstrated in human adipocytes and adipose tissue by Northern blotting and PCR. TNF protein was quantitated by Western blotting and ELISA in both adipose tissue and the medium surrounding adipose tissue. Using quantitative reverse transcriptase PCR (RT-PCR), TNF mRNA levels were examined in the adipose tissue of 39 nondiabetic subjects, spanning a broad range of body mass index (BMI). There was a significant increase in adipose TNF mRNA levels with increasing adiposity. There was a significant correlation between TNF mRNA and percent body fat (r = 0.46, P < 0.05, n = 23). TNF mRNA tended to decrease in very obese subjects, but when subjects with a BMI > 45 kg/m2 were excluded, there was a significant correlation between TNF mRNA and BMI (r = 0.37, P < 0.05, n = 32). In addition, there was a significant decrease in adipose TNF with weight loss. In 11 obese subjects who lost between 14 and 66 kg (mean 34.7 kg, or 26.6% of initial weight), TNF mRNA levels decreased to 58% of initial levels after weight loss (P < 0.005), and TNF protein decreased to 46% of initial levels (P < 0.02). TNF is known to inhibit LPL activity. When fasting adipose LPL activity was measured in these subjects, there was a significant inverse relationship between TNF expression and LPL activity (r = -0.39, P < 0.02, n = 39). With weight loss, LPL activity increased to 411% of initial levels. However, the magnitude of the increase in LPL did not correlate with the decrease in TNF. Thus, TNF is expressed in human adipocytes. TNF is elevated in most obese subjects and is decreased by weight loss. In addition, there is an inverse relationship between TNF and LPL expression. These data suggest that endogenous TNF expression in adipose tissue may help limit obesity in some subjects, perhaps by increasing insulin resistance and decreasing LPL.

Definition of a lamina propria T cell responsive state. Enhanced cytokine responsiveness of T cells stimulated through the CD2 pathway.

This study was designed to compare cytokine release in lamina propria lymphocytes (LPLs) and PBLs activated by Abs against CD3, CD2, and CD28. LPL T cells were significantly more responsive to CD2 ligation than PBL, as determined by release of IFN-gamma, IL-2, IL-4, and TNF-alpha. Moreover, CD28 co-ligation in LPLs exaggerated CD2 > CD3 dominance in cytokine induction. PHA-activated PBLs expressed more CD2 receptors than freshly isolated LPLs, but were less responsive to activation through CD2, indicating that postreceptor pathways in LPL may be adapted specifically to facilitate CD2-mediated cytokine secretion. Antiphosphotyrosine (APT) immunoblotting revealed inducible substrate phosphorylation during CD2, but not CD3, ligation in whole LPLs, as well as LPL-derived T cell lines. PBLs cocultured with an irradiated B cell line, Daudi, and IL-2 for 5 days attained a CD2-dominant cytokine-secretion pattern with identical tyrosine phosphorylation profiles as freshly isolated LPL or LPL T cell lines. PHA-activated PBLs did not produce these tyrosine phosphorylation profiles. This suggests that B lymphocytes in the lamina propria may contribute to a T cell differentiation process in which CD2, possibly by potentiation of its postreceptor pathway, becomes a prominent receptor for induction of cytokine secretion.

Measurement of tumor necrosis factor alpha mRNA in small numbers of cells by quantitative polymerase chain reaction.

We have developed a method to quantitate TNF alpha-mRNA in small numbers of cells by reverse transcription followed by competitive polymerase chain reaction (RT-PCR). RT-PCR allowed the accurate quantitation of TNF alpha-mRNA over a 1000-fold range of concentration. The recovery of RNA isolated from 1000 to 10,000 cells was optimized by reducing sample volumes and by adding 1 microgram yeast RNA. Using these modifications we accurately measured TNF alpha-mRNA in as little as 10,000 U937 cells by RT-PCR. Then we measured TNF alpha-mRNA in lamina propria mononuclear cells isolated from uninflamed and inflamed colonic mucosa from patients with inflammatory bowel disease (IBD). A 9-fold increase (5.4 copies per cell) was found in mononuclear cells from the gut of the inflamed regions compared to those cells from the uninflamed regions (0.6 copies per cell). These findings demonstrated the utility of this method in measuring differences of expression of the TNF-alpha gene in small number of cells isolated from tissues.

Triggered human mucosal T cells release tumour necrosis factor-alpha and interferon-gamma which kill human colonic epithelial cells.

T cell activation can lead to local tissue injury in organ culture studies of human fetal jejunum, either directly through cytotoxicity or indirectly by the release of cytotoxic cytokines. The goal of this study was to establish in vitro whether cytotoxic cytokines can be released by isolated colonic T cells and what cytokine interactions are required for killing of human colonic epithelial cells. Cytokine-containing supernatants were induced by incubating unseparated lamina propria lymphocytes (LPL) or mucosal T cell subpopulations (separated by indirect panning) with anti-CD3 and/or K562 target cells for 18 h at 37 degrees C. Cytokines were measured by cytotoxicity assays using L929 (murine fibroblast) and HT-29 (human colonic tumour) lines as target cells in combination with blocking anti-cytokine antibodies. Supernatants derived from unseparated, CD4+ (greater than 95% pure) and CD8+ (greater than 90% pure) LPL were cytotoxic to L929 targets (350 U/ml, 230 U/ml and 100 U/ml tumour necrosis factor-alpha, respectively). All or nearly all of the cytotoxicity was due to the presence of tumour necrosis factor-alpha (little or no tumour necrosis factor-beta was detected). These same supernatants were cytotoxic (up to 32% lysis at 1/4 dilution) to HT-29 targets in a 48-h 111In release assay. Recombinant tumour necrosis factor-alpha and interferon-gamma alone produced minimal killing of HT-29, but together killed the HT-29 target cells. Anti-tumour necrosis factor-alpha or anti-interferon-gamma alone blocked killing of HT-29 target cells by LPL-derived supernatants, although anti-tumour necrosis factor-beta had no effect upon killing of HT-29. These results demonstrate that human LPL T cells, triggered by addition of anti-CD3 and target cells, produce tumour necrosis factor-alpha and interferon-gamma, both of which are required for optimal killing of HT-29. Simultaneous release of these cytokines in the vicinity of epithelial cells during immune responses could play an important role in the mucosal damage in chronic inflammatory states such as inflammatory bowel disease.

The CD8+ Leu-7+ subset of T cells in Crohn's disease: distinction between cytotoxic and covert suppressor functions.

An expanded T cell subpopulation (CD8+ Leu-7+) has previously been reported in the peripheral blood of patients with Crohn's disease. This subpopulation of T cells was associated with a 'covert suppressor' function, particularly in patients with mild/early Crohn's disease, suppressing immunoglobulin production in vitro when cultured in the presence of pokeweed mitogen. T cells with the same CD8+ Leu-7+ phenotype have also been shown to exhibit non-major histocompatability complex-restricted cytotoxicity when triggered by anti-CD3 antibodies, and this cytotoxic activity has also been shown to be elevated in patients with Crohn's disease. Because cytotoxic cells can have immunoregulatory properties, we investigated the possible relationship between the cytotoxic and 'covert suppressor' functions of the CD8+ Leu-7+ subset of T lymphocytes in patients with mildly active Crohn's disease. Although the correlation between T cell cytotoxic activity and the CD8+ Leu-7+ cells was confirmed, no evidence for covert suppressor activity was found; there were no significant differences between the amount of IgM secreted by B cells from normal subjects and patients with Crohn's disease when cultured with T cells at increasing T:B ratios. In addition, IgM production by peripheral blood B cells did not correlate with either the number of CD8+ Leu-7+ cells or with the level of cytotoxic T cell activity. Furthermore, when B cells and CD4+ T cells were co-cultured with increasing numbers of CD8+ T cells, there was no evidence for excessive suppressor T cell activity in Crohn's disease. Although some patients exhibited low levels of IgM production, this was due to diminished B cell function, rather than excessive T suppressor activity or defective T helper activity. We conclude that the CD8+ Leu-7+ T cell subset is associated with cytotoxic but not with enhanced or covert suppressor activity in Crohn's disease. The previously described covert suppressor function attributed to cells with this phenotype in Crohn's disease was not found to account for diminished B cell responsiveness in vitro and is unlikely to be of major pathophysiologic significance in the majority of patients.

Indoor firing range air quality: results of a facility design survey.

A survey of 611 indoor firing ranges identified features of range design, operation, and maintenance that could affect air quality, a concern because of the lead composition of ammunition fired at the ranges. Features examined included the number of firing positions, location and type of air-handling equipment, maintenance practices, and other standard operating procedures (SOPs). Analysis of the data from the 339 valid responses showed that these ranges vary widely in design, construction, number of firing positions, and frequency of use. Most of these ranges were constructed years ago to the standards in force at the time. Consequently, they do not include many features specified in current standards. Findings from the survey suggest two possible options for ranges concerned about the lead exposure level: design upgrades and SOP changes. Retrofits are costly and design solutions must rely on existing criteria, many of which need verification to ensure their adequacy. Also, more research is needed to define the relationships between ventilation system design and lead exposures at the firing line. A lower cost, more expedient solution is to establish a program of prevention through changes in SOP, such as prohibition of unjacketed lead bullets or establishment of a regular program to monitor the proper operation of ventilation systems. Technology to enhance these preventive measures is being investigated. Possible products include devices for real-time monitoring of ambient air quality and personal lead dosage monitors. This study has underscored the need for further site studies to verify design criteria and to collect chemical and physical data that will help define the nature and extent of problems.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced peripheral blood T-cell cytotoxicity in inflammatory bowel disease.

Monoclonal antibodies to the CD3 component of the T-cell antigen receptor can trigger antigen-specific cytotoxic T cells to elicit nonantigen-specific cytotoxicity, possibly by mimicking or bypassing the requirement for antigen triggering. We have used this technique to investigate the possible presence of in vivo primed cytotoxic T cells, of unknown antigen specificity, in peripheral blood of patients with inflammatory bowel disease. Peripheral blood lymphocytes, which were depleted of background natural killer (NK) activity (CD16-), from patients with Crohn's disease exhibited significantly enhanced levels of anti-CD3-triggered T-cell cytotoxicity compared with lymphocytes from normal subjects. Enhanced lytic activity was also found in some patients with ulcerative colitis and in patients with ulcerative colitis postcolectomy. These results were not influenced by treatment or disease activity. There was no correlation between the anti-CD3-triggered T lytic activity and the NK activity in normal subjects or in patients with inflammatory bowel disease. The surface antigen phenotype of the anti-CD3-triggered T killer cell was CD3+, CD8+, CD16-, and Leu 7+. The results provide indirect evidence for increased activity of a subpopulation of cytotoxic T cells, of unknown antigen specificity, in inflammatory bowel disease. Increased activity in patients with ulcerative colitis postcolectomy suggests that this might reflect a fundamental immunological disturbance.

Role of the CD45 (T-200) molecule in anti-CD3-triggered T cell-mediated cytotoxicity.

NK-depleted human peripheral blood lymphocytes can be modulated with anti-CD3 to kill certain targets during 3-hr cytotoxicity assays. When triggered by anti-CD3 antibody, these effector T cells killed only NK-sensitive targets, such as K562 and HEL 92.1.7, and NK-resistant targets, such as Daudi, whose killing is inhibited by anti-CD45 (T-200) monoclonal antibodies, such as 13.3. NK-sensitive targets, MOLT-4, U266/AF10, Jurkat, and CCFR-CEM, and 10 NK-resistant cell lines, including Raji, IM-9, U698, U937, and GM-1056, whose killing is not inhibited by anti-CD45 monoclonal antibodies, were not killed by alpha-CD3-T effectors, suggesting that the CD45 molecule may be involved in the killing process. Anti-CD3-triggered T cell killing of target cells was inhibited greater than 95% by the monoclonal antibody 13.3. This inhibition of cytotoxicity by 13.3 was not due to competition of this IgG1 antibody for Fc receptor binding site on the target cell, since the IgG1 monoclonal antibody anti-beta 2-microglobulin did not block cytotoxicity. Single cell assays and calcium pulse assays showed that CD45 is involved in a postbinding, pre-calcium-dependent stage, similar to that shown for NK cytotoxicity. There was a relative shift of importance of different epitopes of CD45 in anti-CD3-T cytotoxicity compared to NK cytotoxicity. Anti-CD45 antibodies which bind to the C terminus end of the molecule played a more important role in anti-CD3-T cytotoxicity than NK cytotoxicity. Thus, a subset of T cells exists that exhibits anti-CD3-triggered non-MHC-restricted killing of certain NK-sensitive and NK-resistant targets in association with a CD45 molecule which is functionally different from the NK CD45 molecule.

Human mucosal T-cell cytotoxicity.

Non-major histocompatibility complex-restricted cytotoxicity triggered by antibodies to the CD3 component of the human T-cell receptor complex is thought to be an indirect measure of in vivo primed cytotoxic T-cell activity. We have used this technique to examine the lytic activity of freshly isolated T cells from noninflamed human colonic mucosa. Anti-CD3-triggered T-cell (anti-CD3-T) cytotoxicity was found in all mucosal specimens studied. The mucosal anti-CD3-T effectors do not have Fc receptors for immunoglobulin G, and are therefore distinct from T gamma cells, which mediate antibody-dependent cellular cytotoxicity. The surface antigen phenotype of mucosal anti-CD3-Ts is CD2+, CD3+, CD8+, CD4-, CD16-, and Leu7-. In contrast, peripheral blood anti-CD3-T effectors are Leu7+. Although non-major histocompatibility complex-restricted, mucosal anti-CD3-T cytotoxicity has considerable target specificity, which differs from that of natural killer and lymphokine-activated killer cells. The profile of target cell susceptibility and the inhibitory effects of anti-CD45 antibody suggest that the CD45 molecule on the effector cell may be an important determinant of anti-CD3-T sensitivity. As anti-CD3-triggered lysis may be a marker of in vivo primed mucosal T cells of undetermined antigen specificity, this technique might have important implications in inflammatory bowel disease, where the antigen(s) inciting the mucosal immune reactivity is not certain.

Definition of a secondary target cell trigger during natural killer cell cytotoxicity: possible role of phospholipase A2.

Phospholipase A2 (PA-2) is known to be involved in many calcium-dependent cellular processes and inhibitors of PA-2 have been shown to inhibit natural killer cell-mediated cytotoxicity (NK CMC). Since the trigger stage is calcium dependent, it was postulated that this effector cell-associated enzyme may play a role in early calcium-dependent processes. To define how PA-2 might be involved in NK lysis, the effect of both PA-2 inhibitors and exogenous PA-2 on the stages of NK lysis was examined. PA-2 inhibitors, quinacrine and p-bromophenacyl bromide, inhibited NK CMC at the effector cell level, but affected neither initial target-effector cell binding nor dissociated conjugates during the length of the NK assay, suggesting that they block post-binding lytic events. A calcium pulse assay showed that PA-2 inhibitors inhibit only moderately when added after calcium and only within the first 15 min, demonstrating that these inhibitors blocked very early post-binding lytic events. Because this very early post-binding inhibitory effect was consistent with effects upon the NK trigger mechanism, the effect of exogenous PA-2 on NK lysis was tested. Pretreatment of K562 target cells but not pretreatment of peripheral blood lymphocytes (PBL) with 20 units/ml PA-2 enhanced lysis by two to eight-fold (based upon lytic units), showing its enhancing effect to be at the target cell level. Single cell assays using effector cells purified by indirect panning with monoclonal antibody NKH-1 showed that only the number of killer cells was increased. Calcium pulse assays showed that enhancement of lysis was maximum 15 min after addition of calcium and decreased rapidly thereafter, demonstrating its effect at an early post binding stage. Additionally, PA-2 was shown to overcome inhibition by the monoclonal antibody 13.3, which has been shown to affect the trigger stage of NK lysis (post-binding but prior to calcium dependent events). Thus, it appears that an NK cell-associated PA-2 could function by modulating the target cell surface, revealing a structure which acts as a "secondary" trigger, subsequent to the 13.3 "trigger", requisite for activation of the NK lytic process.

Active target cell processes, possibly involving receptor-mediated endocytosis, are critical for expression of cytotoxicity by natural killer cell-derived cytolytic factor.

Inhibitors of energy metabolism, 2-deoxyglucose and cyanide were shown to inhibit NKCF-mediated lysis of L929 target cells at the same molar concentrations that effectively inhibited cellular ATP levels and the toxic effects of pseudomonas toxin A. In addition, inhibitors of receptor-mediated endocytosis, cytochalasin B, a microtubule disrupter, and trifluoperazine, an inhibitor of clathrin-coat formation, inhibited NKCF-mediated lysis and expression of pseudomonas toxin activity, but had little effect upon cellular ATP. Lysomotropic agents chloroquine, ammonium chloride, and dansylcadavarine also inhibited both NKCF-mediated lysis and pseudomonas toxin activity. These results are similar to those involving diphtheria toxin and the plant toxins abrin, modeccin, and ricin, whose mode of action involves inhibition of protein synthesis following receptor-mediated endocytosis. However, it was determined that NKCF did not cause a decrease in the rate of protein synthesis up to the time of cell death. These results suggest that active target cell processes (possibly involving receptor-mediated endocytosis of NKCF) must occur for target cell lysis to be completed.