Expression of pro-inflammatory cytokines by TCRαβ+ T and TCRγδ+ T cells in an experimental model of colitis

An inflammatory bowel disease (IBD) comparable to human ulcerative colitis is induced upon transfer of T cell-depleted wild-type (F1) bone marrow into syngeneic T cell-deficient (tgε26) mice (F1 → tgε26). Previously we have shown that activated CD4⁺ T cells predominate in transplanted tgε26 mice, and adoptive transfer experiments verified the potential of these cells to cause disease in immunodeficient recipient mice. Using flow cytometry for the detection of intracellular cytokine expression, we demonstrate in the present study that large numbers of CD4⁺ and CD8⁺ TCRαβ⁺ T cells from the intraepithelial region and lamina propria of the colon of diseased, but not from disease-free mice, produced interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Large numbers of T cells from peripheral lymphoid tissues of these animals also expressed IFN-α and TNF-α, but few expressed interleukin-4, demonstrating g strong bias towards Th1-type T cell responses in these animals. TCRγδ⁺ T cells, typically minor constituents of the inflammatory infiltrate of the colon in F1 → tgε26 mice, also expressed IFN-γ at a high frequency upon CD3 stimulation. In light of these findings we examined the potential involvement of TCRγδ⁺ T cells by testing their ability to induce colitis in tgε26 mice. We report here that tgε26 mice transplanted with T cell-depleted bone marrow from TCRα^null and TCRβ^null animals developed IBD. Furthermore, disease in these mice correlated with the development of peripheral and colonic TCRαδ⁺ T cells capable of IFN-γ production. These results suggest that IFN-γ may be a common mediator of IBD utilized by pathogenic T cells of distinct phenotype.     

E-cadherin and α-, β-, and γ-catenin protein expression in relation to metastasis in human breast carcinoma

In the metastatic process, various cell–cell adhesion molecules seem to play an important role. E-cadherin, a transmembrane protein with an extracellular and an intracellular domain, is one of the key players involved in cell–cell adhesion. The function of E-cadherin in preventing metastasis in tumour development is believed to be dependent on intracellular catenins. In a previous study, the expression of E-cadherin was examined in a series of human breast carcinomas. In that study, down-regulation of E-cadherin failed to correlate with lymph node and/or distant metastasis. In the present study, the expression of α-, β-, and γ-catenins has been examined in a subset of the same tumours in order to evaluate their possible role in breast cancer metastasis. Tumour tissues from 90 primary breast carcinomas were immunostained for α-, β-, and γ-catenins. Reduced or absent immunoreactivity in the tumour tissue was seen in 63 (70·0 per cent) for α-catenin, in 50 (55·6 per cent) for β-catenin, and in 50 (55·6 per cent) for γ-catenin. Reduced expression of each of the catenins alone failed to correlate to metastasis. However, when all of the four proteins (E-cadherin, α-catenin, β-catenin, and γ-catenin) were analysed as one group, a significant association was seen between reduction in immunoreactivity of at least one of these four proteins and the presence of metastases. These results indicate that if one of these proteins is down-regulated, the function of the others in suppressing metastasis is altered. A significant association was seen between lobular invasive tumours and β-catenin expression. © 1998 John Wiley & Sons, Ltd.     

β1 and β4 integrin expression in methacarn and formalin-fixed material from in situ ductal carcinoma of the breast

The integrins are αβ heterodimeric transmembrane proteins mediating cell-substratum as well as cell-cell interactions. Previous distribution studies on integrin expression have been limited by the requirement for cryostat sectioned tissues, and consequent poor resolution. We have examined 40 examples of ductal carcinoma in situ (DCIS) for the expression of both β1 and β4 integrin chains. These showed strong polarized membrane staining of residual myoepithelial cells (correlating with expression of smooth muscle specific actin) and of the basement membrane region with β1 and β4 antibodies respectively. In 12 out of 40 cases, the DCIS was negative for the β1 chain and a variable pattern of reactivity was seen in the remaining cases. The β4 chain was detected focally and weakly in the tumour cells of 7/40 DCIS and strongly in one; all of these cases were also positive for the β1 chain. Of the 22 cases where co-existent invasion was present, the infiltrating component showed either a similar degree or a diminution of the extent of immunostaining when compared with the in situ component; only one showed enhanced staining (β1 only). This study demonstrates that two of the main β chains, β1 and β4, can be effectively demonstrated on methacarn and cold (4°C) formalin-fixed tissues by avidin-biotin indirect immunoperoxidase staining and that the results are similar to those achieved using frozen tissue.     

IL-1β TNF-α, and IL-2 in Peritoneal Fluid and Macrophage-Conditioned Media of Women With Endometriosis

PROBLEM : The presence of various cytokines in human peritoneal fluid has been incompletely evaluated. Changes in cytokine levels may be related to the development of endometriosis, infertility, and activation of peritoneal macrophages. This study assesses levels of IL-1β, IL-2, and TNF-α in peritoneal fluid and macrophage conditioned media of women with endometriosis. METHOD : Peritoneal fluid was collected from 51 women at the time of diagnostic or operative laparoscopy for benign gynecologic disease. Peritoneal macrophages were isolated, cultured for 24 h, and the culture media collected. IL-1β, IL-2, and TNF-α levels were determined by commercial ELISA kits. RESULTS : The mean concentration of IL-1β and TNF-α was significantly higher in macrophage conditioned media of patients with endometriosis (P < 0.02). However, there were no significant changes in peritoneal fluid cytokine levels. Peritoneal macrophage concentrations were also higher in patients with endometriosis. CONCLUSION : This study supports the concept that endometriosis is associated with activation of peritoneal macrophages, and a higher concentration of these cells. This activation is reflected by the increased levels of cytokines found in macrophage conditioned media. The absence of significant changes in peritoneal fluid cytokine levels would seem to indicate that the above derangements are not responsible for the development or progression of endometriosis.     

Reduced expression of oestrogen receptor‐β is associated with tumour invasion and metastasis in oestrogen receptor‐α‐negative human papillary thyroid carcinoma

Oestrogens play an important role in the development and progression of papillary thyroid carcinoma (PTC) through oestrogen receptor (ER)‐α and ‐β, which may exert different or even opposing actions in PTC. The roles of ERβ in ERα‐negative PTC are still not clear. This study investigated the expression dynamics of ERβ1 (wild‐type ERβ) and its clinical significance in female ERα‐negative PTC patients. ERβ1 expression was detected in thyroid tissues of 136 female patients diagnosed with PTC. The relationships between ERβ1 expression and clinicopathological/biological factors were also analysed in female ERα‐negative PTC patients. The total score for ERβ1 was significantly lower in female ERα‐negative PTC patients with LNM or ETE when compared to those without LNM or ETE (Z = ?2.923, P = 0.003 and Z = ?3.441, P = 0.001). Accordingly, the total score for ERβ1 was significantly higher in ERα‐negative PTC patients expressing E‐cadherin compared to patients negative for E‐cadherin expression (Z = ?2.636, P = 0.008). The total score was lower in ERα‐negative PTC patients positive for VEGF expression compared to those negative for VEGF expression (Z = ?1.914, P = 0.056). This preliminary study indicates that reduced expression of ERβ1 in female ERα‐negative PTC patients is associated with greater progression of the disease. This may provide insights into the underlying molecular mechanisms of ERβ1 and could help design targeted approaches for treating or even preventing this disease.     

Urinary excretion of prostaglandin F2α and 6-keto-prostaglandin F1α during volume expansion in man

Renal function and the urinary excretion of immunoreactive prostaglandin F_2α (PGF_2α) and 6-keto-prostaglandin F_1α (6-keto-PGF_1α) were investigated during volume expansion (VE) in 9 healthy young adults. The studies were started after at least 17 h of food and fluid deprivation. Volume expansion (3% of body weight) was achieved by a continuous infusion of Ringer's solution (0.22 ml/kg/min). This increased the urinary excretion of sodium from 195±25 to 714±55 μmol/min/1.73 m²(mean ± S.E.) and decreased the excretion of potassium by 24% and plasma renin activity by 60% (P<0.01). The clearance of inulin increased slightly (from 102.4±3.7 to 114.5±6.2 ml/min/1.73 m², P<0.025), whüe clearance of PAH did not change. The excretion of immunoreactive PGF_2α decreased in 8 out of 9 individuals during VE, from 1.58±0.15 to 0.97±0.10 ng/min/1.73 m²(P<0.01). In contrast, excretion of immunoreactive 6-keto-PGF_1α increased in 8 out of 9 subjects, from 2.32±0.20 to 3.47±0.48 ng/min/1.73 m²(P<0.05). Urinary excretion of PGF_2α and 6-keto-PGF_1α may reflect renal synthesis of prostaglandins (PGs) and prostacyclin (PGI₂), respectively. The results indicate that synthesis of PGs is decreased and that of PGI₂ is increased during VE in man. However, no simple relationship could be found between the prostaglandins and the renal functional parameters.     

The expansion and selection of T cell receptor αβ intestinal intraepithelial T cell clones

In conventional mice, the T cell receptor (TCR)αβ⁺ CD8αα⁺ and CD8αβ⁺ subsets of the intestinal intraepithelial lymphocytes (IEL) constitute two subpopulations. Each comprise a few hundred clones expressing apparently random receptor repertoires which are different in individual genetically identical mice (Regnault, A., Cumano, A., Vassalli, P., Guy-Grand, D. and Kourilsky, P., J. Exp. Med. 1994. 180: 1345). We analyzed the repertoire diversity of sorted CD8αα and CD8αβ⁺ IEL populations from the small intestine of individual germ-free mice that contain ten times less TCRαβ⁺ T cells than conventional mice. The TCRβ repertoire of the CD8αα and the CD8αβ IEL populations of germ-free adult mice shows the same degree of oligoclonality as that of conventional mice. These results show that the intestinal microflora is not responsible for the repertoire oligoclonality of TCRαβ⁺ IEL. The presence of the microflora leads to an expansion of clones which arise independently of bacteria. To evaluate the degree of expansion of IEL clones in conventional mice, we went on to measure their clone sizes in vivo by quantitative PCR in the total and in adjacent sections of the small intestine of adult animals. We found that both the CD8αα and the CD8αβ TCRαβ IEL clones have a heterogeneous size pattern, with clones containing from 3 × 10³ cells up to 1.2 × 10⁶ cells, the clones being qualitatively and quantitatively different in individual mice. Cells from a given IEL clone are not evenly distributed throughout the length of the small intestine. The observation that the TCRαβ IEL populations comprise a few hundred clones of very heterogeneous size and distribution suggests that they arise from a limited number of precursors, which may be slowly but continuously renewed, and undergo extensive clonal expansion in the epithelium.     

αβ Lineage-committed thymocytes can be rescued by the γδ T cell receptor (TCR) in the absence of TCR β chain

Commitment of the αβ and γδ T cell lineages within the thymus has been studied in T cell receptor (TCR)-transgenic and TCR mutant murine strains. TCRγδ-transgenic or TCRβ knockout mice, both of which are unable to generate TCRαβ-positive T cells, develop phenotypically αβ-like thymocytes in significant proportions. We provide evidence that in the absence of functional TCRβ protein, the γδTCR can promote the development of αβ-like thymocytes, which, however, do not expand significantly and do not mature into γδ T cells. These results show that commitment to the αβ lineage can be determined independently of the isotype of the TCR, and suggest that αβ versus γδ T cell lineage commitment is principally regulated by mechanisms distinct from TCR-mediated selection. To accommodate our data and those reported previously on the effect of TCRγ and δ gene rearrangements on αβ T cell development, we propose a model in which lineage commitment occurs independently of TCR gene rearrangement.     

The expression and significance of WWOX and β‐catenin in hepatocellular carcinoma

WW domain‐containing oxidoreductase (WWOX) is a novel tumor suppressor gene, and its expression is reduced in various cancers, including hepatocellular carcinoma (HCC). WWOX has been reported to be downregulated in HCC cell lines as well as in primary HCC tissues. It has been suggested that WWOX is implicated in Wnt/β‐catenin pathway, which is frequently affected in HCC. The aim of this study was to evaluate the expression of WWOX, β‐catenin and T‐cell factor 4 (TCF4) in HCC. Our result showed that downregulation of WWOX in HCC was correlated with cytoplasmic accumulation of β‐catenin. In addition, strong nuclear TCF4 expression was associated with tumor grade and stage in HCC. In conclusion, our result implied that downregulation of WWOX might lead to accumulation of cytoplasmic β‐catenin and the subsequent activation of Wnt/β‐catenin signaling pathway in HCC.     

Patterns of integrin common chain β1 and collagen IV immunoreactivity in hepatocellular carcinoma. Correlations with tumour growth rate,grade and size

Thirty cases of hepatocellular carcinoma (HCC) were investigated immunocytochemically for expression of β1 integrin molecule and of collagen IV. Immunoreactivity was related to the tumour proliferation index, as detected by PCNA immunostaining, and to tumour size and grade. Membrane β1 integrin immunoreactivity was deteccted in the neoplasticc cells of all cases, though two different staining patterns were clearly recognized. In 14 cases, β1 integrin immunoreactivity was confined to the cell-stroma interface, showing the same polarized pattern as the non-neoplastic cell counterpart. This staining pattern was associated significantly (P 0.0001) with low PCNA labelling (i.e. less than 20 per cent of neoplastic cells showing nuclear immunostaining. Conversely, 16 cases showed non-polarized pericellular β1 integrin immunostaining. This staining pattern was significantly associated (P < 0.0001)) with high PCNA labelling (more than 20 per cent of immunoreactive cells) and with tumour size greater than 4 cm in diameter (P < 0.0001). β1 Integrin, collagen IV, and PCNA immunoreactivities, however, did not correlate with the histological grade. The data emphasize that neoplastic progression of HCCs may be correlated with an aberrant expression of adhesion molecules and with a disruption of the collagen IV complement of basal membranes.     

Postjunctional α1- and α2-adrenoceptors mediating contraction in isolated human groin arteries and veins

By means of selective agonists and antagonists for α₁- and α₂-receptors, the α-receptor subtypes in human groin arteries and veins were characterized and compared. In the arteries the α₁-receptor blocker prazosin caused a concentration-dependent parallel displacement of the noradrenaline (NA) concentration-response (cr) curve without reduction of maximum (pA₂=9.86); the selective α₂-receptor antagonist rauwolscine in the concentration 10^-8 M caused a right-ward shift of the NA cr-curve without reduction of E_max, but 10^-7 M and 10^-6 M caused little or no further shift. In the veins, the two antagonists had the opposite effects. Rauwolscine caused a concentration-dependent right-ward shift of the NA cr-curve without depression of maximum (pA₂=9.03); prazosin 10^-9 M significantly displaced the NA cr-curve, whereas 10^-8 M and 10^-7 M caused little or no further shift. The responses to the α₂-receptor agonist clonidine in the arteries were too small to allow calculations of pEC50 values; in the veins contractions were elicited in all vessel segments investigated (pEC₅₀=6.24). Phenylephrine, selective for α₁-receptors, was significantly more potent in arteries than in veins. NA was significantly more potent in veins than in arteries. It is concluded that in human groin vessels, there is a functional predominance of arreceptors in the arteries and of a2-receptors in the veins.     

Distribution of α4β7 and αEβ7 integrins on thymocytes,intestinal epithelial lymphocytes and peripheral lymphocytes

β₇ is expressed on subsets of thymocytes, while T and B lymphocytes show heterogeneous expression of β₇. Here, we examine the phenotype of the thymocyte and lymphocyte subsets which express α₄β₇ and α_Eβ₇ using mAb against α_Eβ₇ and mAb DATK32 which recognizes a combinatorial epitope on α₄β₇. β₇⁺ thymocytes have a mature phenotype: TcR⁺, CD11a^hi CD44^hi HSA^dull. Small subsets of double-negative CD4_?CD8_?, single-positive CD4⁺ and CD8⁺ thymocytes express α₇, while double-positive CD4⁺ CD8⁺ thymocytes are β₇. However, two integrins α_Eβ₇ and α₄β₇ recognized by anti-β₇ are not expressed on an identical subpopulation of thymocytes, as β_Eα₇⁺α₄β₇^?, α_Eβ₇⁺α₄β₇⁺ and α_Eβ₇^?α₄β₇⁺ thymocyte subsets are evident. Similarly, intraepithelial lymphocytes express high levels of α_Eβ₇ but little α₄β₇. In the spleen, Peyer's patches and lymph nodes, α₄β₇ is expressed at higher levels on most B lymphocytes than on the majority of T lymphocytes, while a small subset of T lymphocytes, which includes both CD4⁺ and CD8⁺ lymphocytes, express high levels of β₇ in the form of α₄β₇ and α_Eβ₇, although, as observed with lymphocytes, not all α₄β₇^hi CD4^? lymphocytes expressed α₄β₇. The population of α₄β₇^hi CD4 lymphocytes are enriched in Peyer's patches and form subsets of the memory CD4⁺ lymphocyte population, which can be further subdivided on the basis of α_Eβ₇, L-selectin and α₄ expression. Therefore, memory CD4⁺ lymphocytes are highly heterogeneous in their expression of adhesion receptors, and presumably these subpopulations will exhibit very different trafficking properties.     

Interleukin-2 receptor β and γ chain dysregulation during the inhibition of CD4 T cell activation by human immunodeficiency virus-1 gp120

We have observed that CD4 T lymphocytes from human immunodeficiency virus (HIV)-infected patients marginally express interleukin-2 receptor (IL-2R)β and IL-2Rγ chains which are essential for IL-2 signal transduction. To analyze this observation further, we studied the influence of gp120 on the cell surface expression of IL-2Rβ and IL-2Rγ by purified CD4 lymphocytes in vitro. Cross-linking of the T cell receptors of these lymphocytes initiates entry into the cell cycle as measured by CD69 and CD71 cell surface expression and [³H]thymidine incorporation. It also induces the cell surface expression of IL-2Rβ and IL-2Rγ. We have shown that treatment of the CD4 T lymphocytes with HIV-1 gp120 before anti-CD3 stimulation impedes cell cycle progression as measured by reduced CD71 expression and inhibition of [³H]thymidine incorporation. Furthermore, cell surface expression of IL-2Rβ and IL-2Rγ subunits, which form the functional intermediate-affinity IL-2R, are significantly inhibited. More importantly, addition of exogenous IL-2 does not restore the proliferation of the CD4 T cells treated with gp120, suggesting that cells are anergic and/or that the remaining IL-2R are not functional. This is the first study of IL-2Rβ and IL-2Rγ dysregulation in the context of HIV infection and shows that CD4 is also involved in IL-2R expression.