Phenotypic characterization of isolated intraepithelial lymphocytes in patients with ulcerative colitis and normal controls

A method was developed to isolate colonic intraepithelial lymphocytes in patients with ulcerative colitis (N=8) and normal controls (N=13) in order to characterize their phenotype using a panel of monoclonal antibodies and flow cytometry. In both groups, the majority of the cells are of the CD3⁺CD8⁺ phenotype associated with cytotoxic/suppressor function. The CD4/CD8 ratios were similar. Virtually no B cells or macrophages were found. An increase in cells coexpressing the CD3 and HLA-DR molecules and a decrease in natural killer cells (CD3, CD16⁺CD56⁺) were found in ulcerative colitis, but this was not significant. There were no differences in the proportions of CD8⁺ and CD4⁺ cells expressing the Leu8 antigen between ulcerative colitis and controls. Thus a population of colonic intraepithelial lymphocytes has been isolated and, although they may be functionally different in ulcerative colitis compared with controls, this cannot be explained in terms of phenotypic characteristics.     

LFA-1 subunit expression in ulcerative colitis patients

The adhesion molecule, lymphocyte function associated antigen 1 (LFA-1) consisting of two subunits, CD11a and CD18, mediates lymphocyte migration into tissue and cell effector functions. Previous observations showed no differences in LFA-1 expression by circulating lymphocytes between inflammatory bowel disease patients and controls. The aim of the present work was to study subsets of circulating LFA-1⁴ lymphocytes in ulcerative colitis (UC) patients versus healthy controls. Peripheral blood mononuclear cells were obtained from 16 UC patients and 10 healthy volunteers. The percentages of CD11a^lo, CD11a^hi; CD18^lo, CD18^hi T and B cells, as well as CD25 expression on these cells were studied using double staining with monoclonal antibodies and panning procedures. The percentage of CD11^hi and CD18^hi T cells was significantly decreased in quiescent UC patients as compared to active disease patients and healthy controls (P<0.05). The majority of CD25⁺ T cells were expressing CD11a and CD18 with low density. A detectable percentage, 2% (range 1–6%), of CD11a^hiCD25⁺ (but not CD18^hiCD25⁺) was found in UC patients with moderate to severe disease, but not in those with inactive UC or healthy controls. In conclusion, the percentage of CD11a^hi and CD18^hi T cells is decreased in peripheral blood of quiescent UC patients, which is probably associated with the effect of specific treatment. The percentage of CD11a^hi+ T cells is increased in peripheral blood of patients with active (moderate and severe) UC, which most likely reflects a sustained T-cell activation due to a persistent inflammatory process.The project was supported by grants from direktor Jacob Madsen's and Hustru Olga Madsen's Foundation.     

Fas/Fas Ligand Expression and Characteristics of Primed CD45RO+ T Cells in the Inflamed Mucosa of Ulcerative Colitis

Background: Chronic immune activation in the colon is characteristic of ulcerative colitis (UC). Fas/Fas ligand (FasL) system is a mechanism responsible for activation-induced cell death (AICD), which maintains homeostasis within the immune system. Thus, Fas/FasL expression on activated colonic T cells of UC patients, as well as the susceptibility of such T cells to AICD was investigated in order to determine the role of activated colonic T cells in the long lasting inflammation in UC. Methods: Fas, FasL, and CD45RO expression on peripheral blood and colonic T cells of UC patients were assayed by flow cytometry. Apoptosis of colonic T cells induced by anti Fas antibody was assessed using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay. Results: The majority of colonic T cells expressed both CD45RO and Fas in the colonic mucosa, a situation that was quite different from that in the peripheral blood. The number of CD45RO⁺CD8⁺ and Fas⁺CD8⁺ T cells was significantly lower in UC patients than the controls, unlike the number of Fas⁺CD4⁺ T cells. In contrast, the number of both CD45RO⁺CD4⁺ and CD45RO⁺CD8⁺ T cells in UC mucosa expressing FasL was significantly higher than in the controls. While Fas mediated apoptosis of CD45RO⁺CD8⁺ T cells was higher in UC patients than the controls, the number of apoptotic CD45RO⁺CD4⁺ T cells from UC mucosa was not. Conclusions: In UC patients, CD45RO⁺CD4⁺ T cells are less sensitive to apoptotic signals mediated by Fas. These phenomena may contribute to the pathogenesis of UC.     

Expression of CD4+ forkhead box P3 (FOXP3)+ regulatory T cells in inflammatory bowel disease

OBJECTIVE: Forkhead box P3 (FOXP3) plays an important role in the development and function of CD4⁺ regulatory T (Treg) cells. In this study the percentage of CD4⁺ FOXP3⁺ Treg cells in peripheral blood mononuclear cells (PBMC) and the frequency of Treg cells in the colonic mucosa of patients with inflammatory bowel disease (IBD) were investigated. METHODS: The percentage of CD4⁺FOXP3⁺ Treg cells in PBMC was analyzed by flow cytometry. Immunohistochemistry was used to examine the FOXP3⁺ cells in the inflamed mucosa. Real‐time polymerase chain reaction and Western blot were used to detect the expressions of FOXP3 mRNA and protein in PBMC and mucosal biopsy specimens of IBD patients, respectively. RESULTS: Together with the decrease of percentage of Treg cells in PBMC, we found that the frequency of Treg cells increased significantly in inflamed mucosa of active or inactive Crohn's disease (CD) and ulcerative colitis (UC). The expressions of FOXP3 mRNA and protein increased in inflamed mucosa when compared with those in healthy controls, especially the FOXP3 mRNA in patients with active CD or UC. Interestingly, the expression of FOXP3 protein in active UC was higher than that in active CD. CONCLUSIONS: There was a decrease of CD4⁺FOXP3⁺ Treg cells in peripheral blood and an accumulation of Treg cells in inflamed mucosa. These data suggested that the suppressive function of Treg cells may be partially inhibited and this could be an important factor in the recurrence of disease, especially in UC.     

A quantitative immunohistochemical evaluation of inflammatory cells at the affected and unaffected sites of inflammatory bowel disease

Levels of T lymphocytes, histiocytes and mast cells have been reported to be increased in the affected mucosa of Crohn's disease (CD) and ulcerative colitis (UC), but the colorectal distribution of these cells is not fully understood. We hypothesized that differences in cell densities between CD and UC would be characteristic, not only in the affected, but also in the unaffected mucosa. The aims of the present study were to clarify whether there were any differences in cell densities in CD, UC and infectious colitis (IC) in the affected mucosa and between CD and UC in the unaffected mucosa. Using mouse monoclonal antibodies recognizing memory T cells (OPD4), cytotoxic/suppressor T cells (C8/144B), histiocytes (PG-M1) and mast cells (AA1), we evaluated mucosal cell densities in biopsy specimens from both endoscopically affected and unaffected sites of CD (n= 12) and UC (n= 15) and from affected sites of IC (n= 10). Ten normal controls were also examined. At affected sites, all cells were significantly more abundant in UC than in the other conditions, except that the density of PG-M1⁺ in CD was similar to that seen in UC. Although the densities of OPD4⁺ and C8/144B⁺ cells at unaffected sites were slightly higher in both CD and UC and in UC, respectively, there was no significant difference in cell densities between CD and UC. The ratio of OPD4⁺ cell density at affected sites to that at unaffected sites was appreciably higher in UC than in CD. The results suggest that a common feature of UC and CD is an increase in PG-M1⁺ cells at the affected mucosa but that the other inflammatory cells studied are more abundant, particularly in UC, and that the difference between UC and CD is conspicuous when comparing the OPD4⁺ cell density of the affected mucosa with that of the unaffected mucosa.     

Disequilibrium in the CD8<Superscript>+</Superscript>CD28<Superscript>+</Superscript>/CD8<Superscript>+</Superscript>CD28<Superscript>−</Superscript> T Lymphocyte Balance Is Related to Prognosis in Rats with Trinitrobenzenesulfonic Acid-Induced Colitis

Purpose

The CD8⁺CD28⁺/CD8⁺CD28^? T lymphocyte balance is vital for human ulcerative colitis (UC) but has not been defined in experimental colitis. This investigation will try to identify the changes that occur in the CD8⁺CD28⁺/CD8⁺CD28^? T lymphocyte balance during the progression of trinitrobenzenesulfonic acid (TNBS)-induced colitis in rats.

Methods

The frequencies of blood CD8⁺CD28⁺ and CD8⁺CD28^? T lymphocytes were detected in the rats belonging to the normal, model, and treated groups on five days using flow cytometry. The treated rats were administered with mesalazine and were euthanized after a 14-day treatment, as were the normal and model rats. The sensitivity and specificity of the CD8⁺CD28⁺/CD8⁺CD28^? T lymphocyte balance in diagnosing early colitis were analyzed by receiver operating characteristics (ROC) curves. The frequencies of CD8⁺CD28⁺ and CD8⁺CD28^? T lymphocytes in the colon tissue were tested via immunofluorescence. ELISA was used to measure the levels of the cytokines. Immunohistochemistry and Western blotting were used to detect the colonic expression of JAK3, STAT6, NFATc2, and GATA3.

Results

We found that the ratio of CD8⁺CD28⁺/CD8⁺CD28^? T lymphocytes decreased, as did the level of interleukin-7, but not IL-12p40, IL-13, or IL-15, in the blood; however, the ratio increased along with JAK3, STAT6, NFATc2, and GATA3 in the colon of the rats with colitis. The changes were effectively reversed through the administration of mesalazine for 13 days. Surprisingly, the balance in the blood could sensitively distinguish rats with early colitis from normal rats.

Conclusion

These data show that increase in CD8⁺CD28⁺ T cells in blood and decrease in CD8⁺CD28^? T cells in colon are associated with experimental colitis.

     

Diminution of Circulating CD4<Superscript>+</Superscript>CD25<Superscript>high</Superscript> T Cells in Na?ve Crohn’s Disease

Crohn’s disease is considered to be caused either by an excess of T-cell effector functions and/or by a defective regulatory T-cell compartment. The aim of this study was to assess in Crohn’s disease the frequency of circulating CD4⁺CD25^high T cells that possess regulatory T-cell functions and CD4⁺CD25^low T cells that contain activated T cells. Flow cytometry of peripheral blood was used to assess CD4⁺CD25^high and CD4⁺CD25^low T-cell frequencies in a cohort of 66 patients with Crohn’s disease in comparison to 19 patients with ulcerative colitis and 31 healthy individuals enrolled as controls. The CD4⁺CD25^high T-cell frequency was significantly lowered in naïve Crohn’s disease (P = 0.013) and in ulcerative colitis (P = 0.001). CD4⁺CD25^low T-cell frequency was increased in Crohn’s disease (P = 0.0001) and in ulcerative colitis (P = 0.0002). Both CD4⁺CD25^high and CD4⁺CD25^low T-cell frequencies are altered in naïve Crohn’s disease resulting in an imbalance between both populations and a relative contraction of the CD4⁺CD25^high T-cell population.     

Demonstration of Low-Regulatory CD25High+CD4+ and High-Pro-inflammatory CD28−CD4+ T-Cell Subsets in Patients with Ulcerative Colitis: Modified by Selective Granulocyte and Monocyte Adsorption Apheresis

Low-CD25^High+CD4⁺, a subset of regulatory CD25⁺CD4⁺ T cells and high-inflammatory CD28⁻CD4⁺ T cells can exacerbate ulcerative colitis (UC). This study sought to investigate the frequency of CD25^High+CD4⁺ and CD28⁻CD4⁺ T cells in patients with UC and the changes in these cells during Adacolumn granulocyte and monocyte adsorption apheresis (GMA). Subjects were 12 patients with active UC, 11 with quiescent UC, and 14 healthy volunteers (HVs). The mean clinical activity index was 15.7 ± 2.2 in active UC and 4.5 ± 1.1 in quiescent UC. Peripheral blood samples were stained with CD4, CD25, and CD28 antibodies for flow cytometry. Patients with active UC received GMA and blood samples were examined before and after the first GMA session. Patients with active UC (P < 0.04) or quiescent UC (P < 0.02) had a higher percentage of CD28⁻D4⁺T cells compared with HVs, while the percentage of CD28⁺CD4⁺ T cells was lower in both UC groups compared with HVs (P = 0.03 and P < 0.02). Patients with active UC had a lower percentage of CD25^High+CD4⁺T cells compared with quiescent UC patients (P < 0.001). A significant increase in CD25^High+CD4⁺ T cells was associated with GMA (P < 0.03). Low CD25^High+CD4⁺ and high CD28⁻CD4⁺ are prominent features in UC. The increase in CD25^High+CD4⁺ T cells induced by GMA should contribute to improved immune function. Additional studies are warranted, since a low frequency of CD25^High+CD4⁺ ₋ and a high frequency of CD28⁻CD4⁺ ₋ expressing T cells might be a predictor of clinical response to GMA.     

Phenotypic and functional characterization of T-cell lines generated from colonoscopic biopsy specimens in patients with ulcerative colitis

Intestinal T-cell lines were generated from lamina propria mononuclear cells isolated from colonoscopic biopsies in ulcerative colitis patients and controls. In both ulcerative colitis and controls, expanded cells were constituted largely by T-cell receptor ⁺, CD4⁺, CD45RA^– (helper), and CD8⁺, CD11b^– (cytotoxic) phenotypes. T-cell receptor V gene usage was not significantly changed after cell expansion and no difference was observed between ulcerative colitis and controls. Ulcerative colitis cells, especially those derived from the patients with long-standing disease, showed significantly higher levels of cytotoxicity against the target cells, including those of colonic epithelial origin, and enhanced production of tumor necrosis factor- and interferon- after short incubation with anti-CD3 antibody. Generation of T-cell lines from colonoscopic biopsy specimens may be useful for detailed functional characterization of locally infiltrating T cells in ulcerative colitis patients.     

Effects of baicalin in CD4 + CD29 + T cell subsets of ulcerative colitis patients

AIM: To evaluate the role of baicalin in ulcerative colitis (UC) with regard to the CD4⁺CD29⁺ T helper cell, its surface markers and serum inflammatory cytokines.METHODS: Flow cytometry was used to detect the percentage of CD4⁺CD29⁺ cells in patients with UC. Real time polymerase chain reaction was used to detect expression of GATA-3, forkhead box P3, T-box expressed in T cells (T-bet), and retinoic acid-related orphan nuclear hormone receptor C (RORC). Western blotting was used to analyze expression of nuclear factor-κB (NF-κB) p65, phosphorylation of NF-κB (p-NF-κB) p65, STAT4, p-STAT4, STAT6 and p-STAT6. The concentrations of interferon-γ (IFN-γ), interleukin (IL)-4, IL-5, IL-6, IL-10 and TGF-β in serum were determined by ELISA assay.RESULTS: The percentages of CD4⁺CD29⁺ T cells were lower in treatment with 40 and 20 μmol/L baicalin than in the treatment of no baicalin. Treatment with 40 or 20 μmol/L baicalin significantly upregulated expression of IL-4, TGF-β1 and IL-10, increased p-STAT6/STAT6 ratio, but downregulated expression of IFN-γ, IL-5, IL-6, RORC, Foxp3 and T-bet, and decreased ratios of T-bet/GATA-3, p-STAT4/STAT4 and p-NF-κB/NF-κB compared to the treatment of no baicalin.CONCLUSION: The results indicate that baicalin regulates immune balance and relieves the ulcerative colitis-induced inflammation reaction by promoting proliferation of CD4⁺CD29⁺ cells and modulating immunosuppressive pathways.     

Granulocyte and Monocyte Adsorption Apheresis Therapy Modulates Monocyte-Derived Dendritic Cell Function in Patients With Ulcerative Colitis

The aim of this study was to elucidate the molecular mechanisms responsible for the therapeutic effects of granulocyte and monocyte adsorption apheresis (GMA). We investigated the alterations in circulating monocyte subsets and monocyte-derived dendritic cell (moDC) function after GMA therapy in ulcerative colitis (UC) patients. Eighteen patients with UC were enrolled: 14 patients were responders, and 4 patients were non-responders. Peripheral venous blood was obtained within 5 min before and 5 min after GMA therapy. Flow cytometric analysis for monocyte markers (CD14/CD16) was then performed. Monocyte-derived dendritic cells were obtained and alterations in their phenotype were analyzed by flow cytometry. Their function was also analyzed in a mixed lymphocyte reaction assay between allo-naïve T lymphocytes. Flow cytometric analysis for intracellular interferon (IFN)-γ (T-helper 1 cells) and interleukin (IL)-4 (T-helper 2 cells) was then performed for the stimulated T lymphocytes. In patients who responded to GMA, the average numbers of monocytes, especially CD16⁺ monocytes, were significantly decreased after therapy (P < 0.05). In responders, post-GMA moDCs expressed significantly lower CD80 and B7-DC, which are one of the stimulation and maturation markers of dendritic cells, compared to pre-GMA moDCs. CD83, CD86 and human leukocyte antigen-DR also showed a tendency to decrease. In responders, naïve T lymphocytes stimulated with post-GMA moDCs produced significantly less IFN-γ and IL-4 compared to those stimulated with pre-GMA moDCs. The results of our study show that some of the immunosuppressive effects of GMA therapy may be associated with the modulation of monocyte subsets and moDC function.     

Colonic mucosal T lymphocytes in ulcerative colitis: Expression of CD7 antigen in relation to MHC class II (HLA-D) antigens

T-cell subsets and their activation state were examined by double-label immunofluorescence of cryostat tissue sections of the colon from 21 patients with ulcerative colitis (UC) and 30 histologically normal controls. Expression of MHC class I (HLA-A, B, C) and class II (HLA-D) antigens was studied in parallel. In the normal colonic mucosa, the CD4CD8 ratio in the epithelial compartment approximated 11, and in the lamina propria, 2.551. Of the CD8⁺ (cytotoxic/suppressor) subset, approximately half did not express the CD5 pan-T marker in either compartment. Virtually no Leu 8⁺ cells were observed, implying that the CD4⁺ subset consisted of helper, rather than suppressor-inducer cells. Classical markers of T-cell activation (CD25, HLA-D) and proliferation were absent, and strong expression of the CD7 immunostimulation marker was approximately equal in both CD4 and CD8 subsets. The epithelium was uniformly negative for class II antigens, but positive for class I. In UC, there were no significant alterations in CD4CD8 ratios in either compartment, and there were no changes with respect to phenotype of the subsets. In 11 of 19 patients (mainly with total colitis), enterocytes were HLA-D⁺. In this HLA-D⁺ group, there was an increase in the percentage of CD4⁺ cells coexpressing CD7; this difference was significant (P<0.02) in the lamina propria. Increased expression of CD7 was also found by the CD6⁺ T cell subset (P<0.05). These results suggest that class II expression is mediated by immunostimulated T helper cells in UC, with consequences for antigen presentation and maintenance of the chronic inflammatory state.HLA-D is used as a generic term for class II major histocompatibility complex (MHC) gene products (HLA-DR, DP, DQ) unless specified otherwise.     

Adsorptive Depletion of α4 Integrin<Superscript>hi</Superscript>- and CX<Subscript>3</Subscript>CR1<Superscript>hi</Superscript>-Expressing Proinflammatory Monocytes in Patients with Ulcerative Colitis

Background  

Two main functionally distinct monocytes phenotypes are known: the CD14^hiCD16⁻ “classical” and the CD14⁺CD16⁺ “proinflammatory” phenotypes. The latter phenotype is elevated in patients with ulcerative colitis (UC) and is suspected to have a major role in the immunopathogenesis of UC.     

Analysis of T-lymphocyte subpopulations in inflammatory bowel diseases by three-color flow cytometry

In order to better define changes in the relative proportion of peripheral blood T-lymphocyte subpopulations in patients with inflammatory diseases of the bowel, we performed simultaneous three-color fluorescence-activated cytometric (FACS) analysis using fluorophore-conjugated monoclonal antibodies with specificity for CD4, CD8, Leu 8, and CD45RA on 22 normal control subjects, 28 patients with Crohn's disease (CD), 15 patients with ulcerative colitis (UC), and 11 patients with intestinal inflammation secondary to etiologies other than inflammatory bowel disease (NIBD). This staining combination allowed enumeration of distinct T-cell subpopulations as follows: virgin CD4⁺, recall antigen helper T cells, nonspecific B-cell helper T cell, virgin CD8⁺, cytotoxic effector and suppressor effector and recall antigen cytotoxic T cells based on a synthesis of published functional analyses. No differences in the proportion of CD4⁺ or CD8⁺ cells or in the CD4⁺/CD8⁺ ratios were evident when UC and NIBD patients were compared to normal subjects. A significant reduction in the proportion of CD4⁺ cells and an increase in CD8⁺ cells was observed, however, in the CD group. When two-color analysis was performed, several significant differences in the proportions of circulating lymphocytes were seen. Specifically, these included significant increases in the number of CD4⁺, Leu 8^– (P<0.01) cells in all disease groups and an increase in CD4⁺, CD45RA⁺ cells in the NIBD group. Conversely, significant decreases in the proportions of CD8⁺, Leu 8⁺ (P<0.01) cells were evident in the Crohn's disease group. Three-color FACS analysis revealed significant differences in the relative proportions of the defined T-cell subpopulations enumerated in the various groups as compared with the normal controls. These included a decrease in the proportions of Leu 8⁺, CD45RA⁺, (virgin) CD8⁺ T cells (P<0.05) and Leu 8^–, CD45RA⁺, (putative recall antigen helper) CD4⁺ T cells (P<0.01) in all patient groups as compared with normal controls. Conversely, an increase in the proportions of Leu 8^–, CD45RA⁺, (putative suppressor effector) CD8⁺ T cells (P<0.01), and Leu 8^–, CD45RA⁺, (function unknown) CD4⁺ T cells (P<0.05) was seen in all patient groups as compared with normal controls. CD patients but not UC or NIBD patients demonstrated a significant increase in the proportion of Leu 8^–, CD45RA⁺, (putative cytotoxic effector) CD8⁺ T cells (P<0.01). An increase in the ratio of Leu 8^–, CD45RA⁺, CD8⁺ (suppressor effector)/Leu 8⁺, CD45RA^–, CD8⁺ (putative cytotoxic effector) T cells was observed in all of the patient groups, but was most accentuated in those with Crohn's disease. Significant decreases in the ratios of Leu 8^–, CD45RA^–, CD4⁺ (putative nonspecific B cell helper)/Leu 8⁺, CD45RA^–, CD4⁺ (recall antigen helper) T cells and Leu 8⁺, CD45RA^–, CD4⁺ (recall antigen helper)/Leu 8^–, CD45RA⁺, CD4⁺ (function unknown) T cells were observed in all of the disease groups studied as compared with normal controls. These results suggest that the proportions of certain peripheral blood T-cell subpopulations are significantly altered in gastrointestinal inflammatory states. Further analysis of these T-lymphocyte subpopulations in the blood and tissues might provide valuable insights into immunological aberrations in inflammatory bowel diseases and might be of value in distinguishing among inflammatory diseases of the intestine.     

Impaired regulation of natural killer cells in immunoglobulin synthesis by peripheral blood mononuclear cells from patients with ulcerative colitis

Immunoglobulin (Ig) synthesis, natural killer (NK) cell activity, and lymphokine production by peripheral blood mononuclear cells (PBMC) were studied in 34 patients with ulcerative colitis (UC). Levels of Ig produced by PBMC were significantly higher in patients with active UC as compared to controls. However, there were no significant differences in Ig-synthesis between patients with inactive UC and controls. NK cell activity was significantly decreased in patients with active UC as compared to controls, and a significant negative correlation was observed between the level of IgA and NK cell activity in patients with UC. Reconstitution experiments demonstrated that CD56⁺ cells from controls suppressed the levels of IgA, when added to the culture containing a constant number of B cells and CD4⁺ cells. In contrast, CD56⁺ cells from patients with active UC completely lacked the capacity to suppress IgA production. In addition, the activities of interleukin-2 and interferon-γ were significantly decreased in patients with active UC. The present study suggests that immunoregulatory abnormality of NK cells exists in patients with UC and impaired NK cell activity may be related to increased Ig-synthesis observed in these patients.     

Fine T-Cell Subsets in Alcoholics as Determined by the Expression of l-Selectin, Leukocyte Common Antigen, and β-Integrin

Alcoholics admitted to the hospital solely for detoxication have been studied by flow cytometry to evaluate changes in the surface markers of peripheral blood leukocytes. As we have shown previously, such patients have an elevated percentage of CD8^hl lymphocytes that are HLA DR⁺; we now demonstrate that they also have striking alterations in the quantitative relationships of the fine T-cell subsets. Both CD4⁺ and CD8^hl lymphocytes have a sharply reduced percentage of the l -selectin⁺ CD45RA⁺ subset, increased percentages of the CD45RA-subsets, and several other fine subset alterations. The fine subset profile suggests, according to current correlations of phenotype and function, that both CD4⁺ suppressor inducer and CD4-dependent CD8⁺ suppressor effector cells are reduced, whereas other subsets, including CD8⁺ CTL or their precursors, are increased in relative percentages. Some of the phenotypic changes are reversible over the several days following withdrawal. In other results, the percentage of CD8^hl lymphocytes expressing CD11b (β-integrin) is shown to be reciprocal with the percentage expressing l -selectin both in normals and alcoholics. However, the regression function of CD11b vs. l -selectin on CD8^hl cells is different for the alcoholics than for the normals, indicating an abnormality in the regulation of the expression of these two adhesion markers. Taken together, this abnormality of adhesion molecules and the fine subset alterations previously described indicate widespread changes in the peripheral lymphocytes of currently drinking alcoholics. These changes suggest functional deficiencies that may include alterations of lymphocyte traffic and other adhesion-dependent functions, and a shift in the balance of regulatory interactions.     

Higher proliferative capacity of T lymphocytes from patients with Crohn disease than from ulcerative colitis is disclosed by use of Herpesvirus saimiri-transformed T-cell lines

Background: T lymphocytes play a crucial role in the pathogenesis of inflammatory bowel disease. Achieving stable T-cell lines, rather than continuous bleeding of patients, is desirable in order to dissect their implication in the disease. Methods: Long-lasting T-cell lines from patients with Crohn disease and ulcerative colitis and from healthy volunteers have been obtained by transformation of T lymphocytes using the lymphotropic Herpesvirus saimiri. Lines were subjected to phenotypic and functional analyses, and the results compared with freshly isolated peripheral blood mononuclear cells. Results: Fresh cells revealed only minor differences between patients and controls, with regard to phenotype and proliferative capacity. In contrast, the use of T-cell lines showed that cells from Crohn disease patients, but not ulcerative colitis patients, over-responded to several membrane or cytoplasmic stimuli when compared to control T-cell lines. Thus, higher responses were found when stimulated with αCD3 and IL2, αCD3 and αCD28, IL2 alone, phorbol esters (PMA) and αCD3 and, finally, PMA and αCD2 (P?&;lt;?0.05 in all instances). Further, lines from patients with Crohn disease responded more vigorously to αCD3 and αCD28 or αCD3 and PMA when compared to ulcerative colitis (P?&;lt;?0.05 in both instances). Conclusions: The data obtained with these lines suggest that T cells from patients with Crohn disease differ in vivo in their proliferative capacity, as compared with those from ulcerative colitis patients, a finding that may reflect the clear Th-1 phenotype found in the former and absent in the latter.     

Effect of oral hepatocyte growth factor gene mediated by attenuated salmonella on 2-, 4-, 6-trinitro-benzene-sulfonic-acid-induced ulcerative colitis in rat

Background and Aim: In order to explore a new therapeutic method, we investigated the effects of exogenously expressed hepatocyte growth factor mediated by attenuated salmonella (TPH) on rats with ulcerative colitis (UC) induced by 2‐, 4‐, 6‐trinitro‐benzene‐sulfonic acid. Methods: The UC rats were treated with TPH, attenuated salmonella with a eukaryotic expression vector (TP) or sodium bicarbonate (model control [MC]) every other day. Cluster of differentiation (CD)4⁺ and CD8⁺ T cells and immunoglobulins in the blood were analyzed by flow cytometry. The HGF expression was determined by immunohistochemistry. A macroscopic‐scale observation of the colon and a histological assessment were also carried out. Results: The CD4⁺T counts and the CD4⁺/CD8⁺ ratio in the TPH group were significantly lower than that in the MC group. The immunoglobulin M and immunoglobulin G₁ levels in the TPH group were significantly lower than that in the MC group and TP group. After treatment with TPH, the symptoms of the ulcerative rats were significantly alleviated. The colonic lesion grades in the TPH group were lower than that in the TP group and MC group. Significant improvement occurred after the TPH treatment, as evidenced by alleviated mucosal inflammation. At 7 days post‐treatment, the HGF expression in the colonic tissues that were treated with TPH was stronger than that in the samples treated with TP. Conclusions: TPH inhibits the proliferation of T lymphocytes and the antibody production of B lymphocytes. Furthermore, it ameliorates mucosal inflammation and promotes the regeneration of mucosa and the healing of the colonic ulceration.