Cd1d-restricted cellular lysis by peripheral blood lymphocytes: relevance to the inflammatory bowel diseases.

The CD1d molecule has been implicated to play a role in inflammatory bowel diseases (IBD), possibly through its presentation of an intestinal antigen trigger. To understand the role of the CD1d class I-like protein in IBD, we investigated the molecule's expression in diseased intestinal tissue and determined its potential to undergo specific recognition by intraepithelial and peripheral blood lymphocytes (PBLs) derived from IBD patients. We have observed an increase in precipitable CD1d in inflamed tissues, which suggests CD1d up-regulation in IBD; this was not accompanied by the occurrence of CD1d-specific cytotoxicity by lymphocytes isolated from the same tissue sites. In contrast, we have observed CD1d-specific cytotoxicity by PBLs from both patients and normal controls mediated by a possibly unique type of lymphocytic cell. These observations support a model in which intestinal inflammation may be initiated by circulating PBLs following the tissue-specific upregulation of CD1d. These activated PBLs may then be the source of the extraintestinal manifestations observed with IBD. We therefore propose that the cells responsible for this activity may play a role in regulating immune responses through the specific recognition of CD1d-specific antigen(s).

Absence of GNAI2 codon 179 oncogene mutations in inflammatory bowel disease.

The human GNAI2 gene coding for G protein, Galphai2, is located on chromosome 3p21 in proximity to the region where an inflammatory bowel disease (IBD) locus has been suggested. Galphai2-deficient mice develop a lethal diffuse colitis that resembles human ulcerative colitis (UC) and frequently progresses to colon adenocarcinoma. Furthermore, the human GNAI2 gene is subject to point mutations at certain positions, including three at codon 179, all of which have been reported in human endocrine tumors. In order to evaluate the possible involvement of this gene in IBD pathogenesis, we have examined GNAI2 codon 179 sequences in 28 familial IBD patients, including 13 UC, 15 Crohn's disease (CD), and 7 patients with colon cancer/dysplasia, from 12 multiplex IBD families. The wildtype codon 179, CGC for arginine, plus the first G of the codon 180 engender a sequence recognizable by the enzyme BstUI. Mutations, therefore, can result in the abrogation of BstUI digestion of polymerase chain reaction (PCR) products containing the codon 179. Using the PCR-restriction fragment length polymorphism technique, all 28 IBD patients, including those with colon cancer, and 14 non-IBD family members show a BstUI-cleavable PCR-banding pattern indicating the presence of wildtype codon 179. We conclude that, in the familial IBD and colon cancer/dysplasia patients studied, there is no detectable mutation in the codon 179 of the GNAI2 gene.

Bowel permeability is improved in Crohn's disease after ileocolectomy.

PURPOSE: Numerous investigators have shown increased bowel permeability in patients with Crohn's disease. It is unclear whether this is a precondition affecting the entire intestine or a consequence of the inflammation and, therefore, only affecting the diseased bowel. The present study tested the hypothesis that resection of diseased bowel in patients with ileocolonic Crohn's disease would correct abnormalities in bowel permeability. METHODS: Ten patients (5 females; mean age, 33 +/- 2 years) with ileocolonic Crohn's disease who underwent elective ileocolic resections had bowel permeability measured preoperatively and postoperatively by the relative urinary clearance of orally consumed lactulose and rhamnose. RESULTS: Mean preoperative bowel permeability in patients with Crohn's disease was significantly elevated relative to healthy volunteers (0.172 +/- 0.04 vs. 0.046 +/- 0.01; P < 0.05, unpaired t-test). After ileocolectomy, bowel permeability decreased in patients with Crohn's disease and reached a normal range by postoperative day 30. CONCLUSIONS: Bowel permeability is increased in patients with ileocolic Crohn's disease because of the presence of diseased bowel and decreases to a normal range in these patients after resection of gross intestinal disease. This suggests that bowel permeability may be a quantitative and clinically effective method with which to assess the presence and severity of diseased bowel in patients with Crohn's disease.

Increased LFA-1 expression in intestines of rats with GVHD after small bowel transplantation.

Experimental graft-versus-host disease (GVHD) causes immune-mediated intestinal injury. The adhesion molecule lymphocyte function associated antigen-1 (LFA-1) is involved in leukocyte homing to areas of inflammatory injury. Our hypothesis was that LFA-1 is increased in the GVHD injured small bowel and colon. We found that animals with GVHD caused by auxiliary small bowel transplantation displayed significantly increased expression of intestinal LFA-1alpha at times of clinical illness when compared to controls. The staining pattern progressed from a few discretely stained cells in the lamina propria on day 5 to diffuse confluent staining of lamina propria on day 13 and was statistically significantly increased from controls at days 10 and 13. CyA-treated animals had intermediate staining between control and day 13 GVHD animals. There was no difference between sham-operated control animals and SBTx animals with GVHD in the amount of staining for LFA-1 in extraintestinal organs normally affected by GVHD. We conclude that: (1) LFA-1 expression in the small bowel and colon progressively increased during GVHD after SBTx; and (2) CyA treatment is associated with decreased LFA-1 expression in the small bowel and colon of GVHD animals after SBTx. LFA-1 may play an important role in immune-mediated injury of the intestine.

Monoclonal antibody to lymphocyte function associated antigen-1 improves graft-versus-host disease.

UNLABELLED: We previously showed that intestinal tissue expression of lymphocyte function associated antigen-1 is increased in animals with graft-versus-host disease after small-bowel transplantation. HYPOTHESIS: Treatment of rats with monoclonal antibody to lymphocyte function associated antigen-1 after small-bowel transplantation will lessen the severity of graft-versus-host disease. METHODS: Graft-versus-host disease was created in Lewis X Brown-Norway F1 rats by heterotopic vascularized small-bowel transplantation from Lewis donors. Transplanted rats were treated with either saline or various regimens of monoclonal antibody to lymphocyte function associated antigen-1. Clinical characteristics, weight loss, spleen index, white blood cell counts, native intestinal histology, bowel permeability, and survival were then compared between groups and appropriate sham-operated and lymphocyte function associated antigen-1-treated controls. RESULTS: Lymphocyte function associated antigen-1-treated rats lost less weight, had larger spleen indexes, more normal white blood cell counts, more normal native intestinal histology, less alteration in bowel permeability, and longer survival than untreated small-bowel transplantation rats. CONCLUSIONS: In this model of graft-versus-host disease after small-bowel transplantation, monoclonal antibody to lymphocyte function associated antigen-1 treatment decreased the severity of graft-versus-host disease and prolonged rat survival.

Amelioration of graft versus host disease with anti-ICAM-1 therapy.

We have previously demonstrated an increase in the adhesion molecule lymphocyte function-associated antigen-1 (LFA-1) in GVHD after small bowel transplantation (SBTx) and a therapeutic effect for the monoclonal antibody (MoAb) to LFA-1 in the same model. The present study evaluated the role of MoAb to LFA-1's ligand, intercellular adhesion molecule-1 (ICAM-1) in GVHD. Methods. GVHD was created in LBNF1 rats by heterotopic vascularized SBTx from Lewis donors. Saline treated SBTx-GVHD and sham-operated control animals were compared to animal groups treated with MoAb to ICAM-1 or MoAb to ICAM-1 and LFA-1. GVHD was evaluated by measuring spleen index, white blood cell count, bowel permeability, weight loss, and animal survival. RESULTS. Animals treated with the MoAb to ICAM-1 appeared clinically to have almost as severe GVHD as untreated animals; however, they had improved spleen indices, less neutropenia and weight loss, and survived longer than untreated animals (range 15-22 days in treated animals vs 12-16 days in untreated animals, P < 0. 01). Treatment with MoAb to both ICAM-1 and LFA-1 appeared to have a synergistic beneficial effect on GVHD (range 19-29 days, P < 0.001 vs untreated animals). Conclusion. MoAb to ICAM-1 alone or in combination with MoAb to LFA-1 ameliorates GHVD after SBTx and prolongs survival.

Decreased expression of LFA-1 on peripheral blood lymphocytes in graft versus host disease.

We have previously demonstrated an increase in lymphocyte function associated antigen-1 (LFA-1) expression in the native intestines of animals with graft versus host disease (GVHD) after small bowel transplantation (SBTx). The present study evaluated LFA-1 expression on peripheral blood lymphocytes (PBLs) during GVHD. GVHD was created in LBNF1 rats by heterotopic vascularized SBTx from Lewis donors and compared to sham-op controls and cyclosporine-treated SBTx rats (SBTx-CyA, 10 mg/kg/day). PBLs were harvested on Postop Day 13 when signs of severe GVHD were present and PBLs were stained for LFA-1, CD3 (T-cell marker), and CD45 (B cell marker) and analyzed on an Epics 5 flow cytometer. Mesenteric lymph node (MLN) lymphocytes from the native intestines were also harvested for each group and stained for LFA-1. There were significant decreases in LFA-1, CD3, and CD45 PBL expression in rats with GVHD. CyA treatment corrected the abnormal CD3 and CD45 ratios, but not LFA-1 expression. It is concluded that: (1) The proportion of PBLs expressing LFA-1 is depressed in animals with clinical GVHD consistent with their recruitment into the host's inflamed tissues. (2) CyA treatment corrects the abnormalities in T and B cell ratios but not the decreased expression of LFA-1 on PBLs and may relate to its known imperfect ability to treat GVHD.

Urokinase does not upregulate the vascular endothelial cell-mediated inflammatory response.

PURPOSE: Urokinase is used clinically for thrombolysis, but little is known of its direct effect on vascular endothelial cells. The following experiments were preformed to assess the in vitro effects of urokinase on vascular endothelial cell growth, adhesion molecule expression, and interaction with lymphocytes, polymorphonuclear leukocytes, and platelets. METHODS: Commercially available human umbilical vein endothelial cells (HUVEC) were cultured with varying concentrations of urokinase (0 to 10,000 IU/ml) (clinical dosage, < or = 500 IU/ml). HUVEC viability was determined from 1 to 4 days. HUVECs were incubated with urokinase (0 to 2000 IU/ml) from 4 to 72 hours. Adherence of 51-chromium-labeled polymorphonuclear leukocytes, platelets, or lymphocytes was then quantitated. In separate experiments HUVEC adhesion molecule expression (intercellular adhesion molecule-1, vascular cell adhesion molecule-1, or endothelial leukocyte adhesion molecule-1) was determined by flow cytometry. RESULTS: There was a decrease of HUVEC viability at suprapharmacologic urokinase concentrations of > or = 2000 IU/ml compared with nontreated control samples (0 IU/ml, 73% +/- 2%, 2000 IU/ml, 60.5% +/- 1.9%, p < 0.05) presumably because of drug toxicity. There was no significantly increased polymorphonuclear leukocyte, lymphocyte, or platelet adhesion to urokinase-treated HUVEC monolayes at any time point. This was also true for each adhesion molecule tested. CONCLUSIONS: Urokinase at clinically relevant concentrations (< or = 500 IU/ml) did not affect endothelial cell viability or growth, nor did it upregulate adhesion molecule expression or cellular adhesion associated with the cell vascular inflammatory response. It is therefore implied that the use of urokinase in vivo similarly would not initiate the vascular inflammatory response.

Awake epidural anesthesia is associated with improved natural killer cell cytotoxicity and a reduced stress response.

BACKGROUND: Laparotomy under general anesthesia is associated with depressed natural killer cell cytotoxicity (NKCC) and compromised clearance of tumor cells. We tested the hypothesis that awake epidural anesthesia (AEA) improves NKCC compared to conventional general endotracheal anesthesia (GEA). PATIENTS AND METHODS: Preoperative, perioperative, and postoperative (day 3) NKCC, plasma epinephrine, norepinephrine, cortisol levels, and 24-hour urinary cortisol levels were measured in 20 patients undergoing open colectomy under either AEA or GEA. RESULTS: Preoperative and postoperative measurements were not significantly different in the two groups. Patients receiving GEA had a significant reduction in NKCC from 36% +/- 4% preoperatively to 22% +/- 4% perioperatively (P = 0.02). Patients receiving AEA had no significant change in NKCC. Perioperative plasma epinephrine and cortisol levels were higher with GEA than AEA. The perioperative 24-hour urinary cortisol excretion values were significantly higher in the group receiving GEA, suggesting a greater stress hormone response in this group compared to AEA patients. CONCLUSIONS: Compared to GEA, AEA appears to preserve perioperative NKCC. This effect may be related to an attenuated stress hormone response associated with AEA. Cancer patients may have improved killing of embolized tumor cells during surgery performed under AEA.

Transforming growth factor-beta 1 serves as an autocrine inhibitor of human endothelial cell/lymphocyte adhesion.

Other investigators have shown that exogenously administered transforming growth factor-beta (TGF-beta) inhibits lymphocyte adherence to vascular endothelial cells (VEC). We examined the role of TGF-beta 1 as an autocrine mediator of lymphocyte adhesion to adult human VECs. VECs were harvested from eight saphenous or cadaveric iliac veins using 0.2% collagenase. Low-passage VECs in MCDB + 0.1% BSA were pretreated for 24 hr with monoclonal anti-TGF-beta 1 antibody (5 micrograms/ml), LPS (5 micrograms/ml), or IL-1 (10 U/ml). Adherence of fluorescently labeled lymphocytes to pretreated VECs was quantitated and results were expressed as relative adhesion compared to untreated control. Total mRNA from LPS- or IL-1-treated VECs was subjected to Northern analysis to determine relative TGF-beta 1 expression. Total TGF-beta 1 protein concentration in supernatants from LPS- or IL-1-treated VECs was determined by ELISA. Data (means +/- SEM) were analyzed by ANOVA with a Newman-Keuls posttest. Neutralizing endogenous TGF-beta 1 with anti-TGF-beta 1 antibody significantly increased adhesion of lymphocytes to VEC monolayers compared to control (125 +/- 3 vs 101 +/- 2%, P < 0.01, n = 8). The level of adhesion was equivalent to that seen with IL-1 stimulation (131 +/- 6%). Spearman correlation of lymphocyte adherence to IL-1- or LPS-treated VECs vs TGF-beta 1 mRNA expression or vs relative TGF-beta 1 protein concentration showed significant inverse relationships (r = -0.82, P < 0.001, and r = -0.87, P < 0.001, respectively). Endogenous TGF-beta 1's inhibitory effect on lymphocyte adhesion was blocked by a specific neutralizing antibody. VEC TGF-beta 1 mRNA expression and TGF-beta 1 production were inversely proportional to lymphocyte adhesion, suggesting down-regulation of TGF-beta 1 in response to proinflammatory cytokines. Together, these observations support the hypothesis that TGF-beta 1 has an autocrine inhibitory role in regulation of lymphocyte adhesion to VECs.

Cimetidine reduces cyclosporine inhibition of interleukin-2 production.

Cimetidine has been shown to up-regulate proliferative and cytotoxic immune responses, which are mediated in part by an increase in interleukin-2 (IL-2) production. Cyclosporine achieves its immunosuppressive effect mainly through inhibition of IL-2 production. A recent clinical report of renal allograft recipients with elevated serum creatinine levels while on a histamine type-2 receptor antagonist raised concern whether the cimetidine increase in IL-2 production was counterbalancing the cyclosporine inhibition of IL-2 and thereby increasing alloreactivity to the transplant. We therefore measured IL-2 production of mitogen-stimulated murine spleen cells with or without cimetidine and cyclosporine and in combination of these two drugs. Cimetidine (10(-4) and 10(-5) M) completely reversed the cyclosporine (0.1 ng/ml and 1 ng/ml)-induced 25 and 41% inhibitions of IL-2, respectively. Cyclosporine (10 ng/ml) reduced IL-2 by 64% and cimetidine partially reversed this inhibition to 48%. All cimetidine groups which reduced the cyclosporine effect on IL-2 were statistically significant (P less than 0.05). These data raise concern about the safety of giving histamine type-2 receptor antagonists to allograft recipients without increasing alloreactivity due to a relative increase in IL-2.

Histamine type-2 receptor antagonist immune modulation. II. Cimetidine and ranitidine increase interleukin-2 production.

The effect of cimetidine and ranitidine (histamine type-2 receptor antagonists) on the production of interleukin-2 (IL-2) by mitogen-activated, normal murine spleen cells was studied in vitro. Cimetidine (10(-4) mol/L to 10(-6) mol/L) increased IL-2 production to a maximal 8.8 +/- 1.6 U (IL-2 activity), as compared with media controls of 1 U. Ranitidine (10(-4) mol/L to 10(-6) mol/L) also increased IL-2 production to a maximal 5.6 +/- 1.2 U, as compared with media controls of 1 U. The increases for both drugs were statistically significant- (p at least less than 0.03 for all doses tested). These data suggest that our previously demonstrated immunofacilitation of proliferative and cytotoxic lymphocyte responses by cimetidine was probably mediated by the presence of increased IL-2. These data further suggest that histamine type-2 receptor antagonists may have immunorestorative potential in clinical immunotherapy of IL-2 deficient states.

Cyclosporine-induced adenosine triphosphate depletion in murine T and B lymphocytes.

Adenosine metabolism in C57BL/6 mouse spleen cells was studied. Adenosine triphosphate (ATP) levels in resting T cells were 26.9 +/- 3.4 ng/10(5) cells compared with 16.5 +/- 3.1 ng/10(5) cells in resting B cells. Cyclosporine (CSA) caused a prompt and severe ATP depletion in both T and B cells, which could be mitigated by the addition of adenosine. B cell ATP levels were returned to normal while T cell levels were only partially restored. The adenosine deaminase inhibitor erythro-9-(2 hydroxy-3 nonyl) adenine (EHNA) also caused ATP depletion in T and B cells, which could similarly be prevented in part by the addition of adenosine. However, when CSA and EHNA were combined, adenosine could no longer protect ATP pools and severe ATP depletion in T and B cells occurred. This suggests that CSA and EHNA affect different steps in the conversion of adenosine to ATP. Although both T and B cell ATP levels were affected by CSA, the ability of supplementary substrate to restore ATP levels to normal in B cells but not in T cells may explain the apparent selective effect of CSA impairing T cell functions with sparing of B cell functions. Furthermore, if causing ATP depletion is associated with immunosuppressive activity, EHNA may be useful in potentiating the immunosuppressive effects of CSA.