The High Affinity IgE Receptor FcεRI Is Expressed by Human Intestinal Epithelial Cells

IgE antibodies play a paramount role in the pathogenesis of various intestinal disorders. To gain insights in IgE-mediated pathophysiology of the gut, we investigated the expression of the high affinity IgE receptor Fc epsilonRI in human intestinal epithelium.Fc epsilonRI alpha-chain, as detected by immunohistochemistry, was positive in epithelial cells for eight of eleven (8/11) specimens from colon cancer patients and 5/11 patients with inflammation of the enteric mucosa. The Fc epsilonRIalpha positive epithelial cells co-expressed Fc epsilonRIgamma, whereas with one exception, none of the samples was positive for the beta-chain in the epithelial layer. The functionality of Fc epsilonRI was confirmed in situ by human IgE binding. In experiments with human intestinal tumor cell lines, subconfluent Caco-2/TC7 and HCT-8 cells were found to express the alpha- and gamma-chains of Fc epsilonRI and to bind IgE, whereas confluent cells were negative for gamma-chains.Our data provide the first evidence that the components of a functional Fc epsilonRI are in vitro expressed by the human intestinal epithelial cells depending on differentiation and, more importantly, in situ in epithelia of patients with colon cancer or gastrointestinal inflammations. Thus, a contribution of Fc epsilonRI either to immunosurveillance or pathophysiology of the intestinal epithelium is suggested

Clone-specific expression, transcriptional regulation, and action of interleukin-6 in human colon carcinoma cells

<p>Abstract</p> <p>Background</p> <p>Many cancer cells produce interleukin-6 (IL-6), a cytokine that plays a role in growth stimulation, metastasis, and angiogenesis of secondary tumours in a variety of malignancies, including colorectal cancer. Effectiveness of IL-6 in this respect may depend on the quantity of basal and inducible IL-6 expressed as the tumour progresses through stages of malignancy. We therefore have evaluated the effect of <it>IL-6 </it>modulators, i.e. IL-1β, prostaglandin E₂, 17β-estradiol, and 1,25-dihydroxyvitamin D₃, on expression and synthesis of the cytokine at different stages of tumour progression.</p> <p>Methods</p> <p>We utilized cultures of the human colon carcinoma cell clones Caco-2/AQ, COGA-1A and COGA-13, all of which expressed differentiation and proliferation markers typical of distinct stages of tumour progression. IL-6 mRNA and protein levels were assayed by RT-PCR and ELISA, respectively. DNA sequencing was utilized to detect polymorphisms in the <it>IL-6 </it>gene promoter.</p> <p>Results</p> <p><it>IL-6 </it>mRNA and protein concentrations were low in well and moderately differentiated Caco-2/AQ and COGA-1A cells, but were high in poorly differentiated COGA-13 cells. Addition of IL-1β (5 ng/ml) to a COGA-13 culture raised IL-6 production approximately thousandfold via a prostaglandin-independent mechanism. Addition of 17β-estradiol (10^-7M) reduced basal IL-6 production by one-third, but IL-1β-inducible IL-6 was unaffected. Search for polymorphisms in the <it>IL-6 </it>promoter revealed the presence of a single haplotype, i.e., -597A/-572G/-174C, in COGA-13 cells, which is associated with a high degree of transcriptional activity of the <it>IL-6 </it>gene. IL-6 blocked differentiation only in Caco-2/AQ cells and stimulated mitosis through up-regulation of c-<it>myc </it>proto-oncogene expression. These effects were inhibited by 10^-8M 1,25-dihydroxyvitamin D₃.</p> <p>Conclusion</p> <p>In human colon carcinoma cells derived from well and moderately differentiated tumours, IL-6 expression is low and only marginally affected, if at all, by PGE₂, 1,25-dihydroxyvitamin D₃, and 17β-estradiol. However, IL-6 is highly abundant in undifferentiated tumour cells and is effectively stimulated by IL-1β. In case of overexpression of an <it>IL-6 </it>gene variant with extreme sensitivity to IL-1β, massive release of the cytokine from undifferentiated tumour cells may accelerate progression towards malignancy by paracrine action on more differentiated tumour cells with a still functioning proliferative IL-6 signalling pathway.</p

(A) Effect of hIL-6 on growth rate of confluent Caco-2/AQ, COGA-1A, and COGA-13 cells

<p><b>Copyright information:</b></p><p>Taken from "Clone-specific expression, transcriptional regulation, and action of interleukin-6 in human colon carcinoma cells"</p><p>http://www.biomedcentral.com/1471-2407/8/13</p><p>BMC Cancer 2008;8():13-13.</p><p>Published online 18 Jan 2008</p><p>PMCID:PMC2257953.</p><p></p> Cellular proliferation was evaluated from [H]thymidine incorporation into DNA after 72 h incubation with rhIL-6 (0–100 ng/ml). Data are means ± SD (= 4 – 16) and expressed as multiples of IL-6-free controls. Statistically significant differences from controls: **, < 0.01; ***, < 0.001 (Student's -test). (B) Time-course of mRNA expression in confluent Caco-2/AQ, COGA-1A, and COGA-13 clones during incubation with 100 ng/ml rhIL-6. Expression of the epithelial cell marker CK8 is shown for comparison. (C) Densitometric evaluation of expression of in relation to CK8 as shown in (B): basal expression ratios in zero time controls were set to 1

Basal and stimulated expression of IL-6 mRNA and protein in human colon carcinoma cell clones

<p><b>Copyright information:</b></p><p>Taken from "Clone-specific expression, transcriptional regulation, and action of interleukin-6 in human colon carcinoma cells"</p><p>http://www.biomedcentral.com/1471-2407/8/13</p><p>BMC Cancer 2008;8():13-13.</p><p>Published online 18 Jan 2008</p><p>PMCID:PMC2257953.</p><p></p> Additions to the culture medium were: 10M indomethacin, 10M PGE, 10M 1,25-(OH)D, 10M 17β-E, 5 ng/ml IL-1β. Upper part: Representative RT-PCR amplifications of mRNA transcripts specific for (culture time 4 h). Expression of epithelial cell marker CK8 is shown for comparison. Lower part: IL-6 release into medium during 24 h culture period. Data are means ± SD, ≥ 4. Note that changes in IL-6 secretion by COGA-13 cells are given on a logarithmic scale. Statistically significant differences: *, < 0.05; **, < 0.01; ***, < 0.001 (Student's -test)

Effect of 1,25-(OH)D(10M) on IL-6-related proliferation of confluent Caco-2/AQ cells

<p><b>Copyright information:</b></p><p>Taken from "Clone-specific expression, transcriptional regulation, and action of interleukin-6 in human colon carcinoma cells"</p><p>http://www.biomedcentral.com/1471-2407/8/13</p><p>BMC Cancer 2008;8():13-13.</p><p>Published online 18 Jan 2008</p><p>PMCID:PMC2257953.</p><p></p> Culture time was 72 h. Cell growth was assayed by [H]thymidine incorporation into cellular DNA (normalised to total protein). Data are expressed as means ± SD, = 4 – 7. Statistically significant differences from controls: *, < 0.05; **, < 0.01 (Student's -test)

Detection of EpCAM positive and negative circulating tumor cells in metastatic breast cancer patients

Background. Immunomagnetic EpCAM based methods are used to enrich circulating tumor cells (CTCs) in metastatic breast cancer (mBC) patients. EpCAM negative CTCs may be missed. We addressed the question of the reliability of an EpCAM dependent assay to enrich CTCs. Methods. To elucidate this issue, our study has been designed to assess two different CTC enrichment technologies (i) in EpCAM positive (+) and EpCAM negative cell lines and (ii) in mBC patients in dependency on their respective EpCAM expression. These two technologies encompass one anti-EpCAM immunomagnetic enrichment technology, MACS HEA MicroBeads® (MACS), and one EpCAM independent density centrifugation method, OncoQuick® plus (OQ+). Furthermore, the coherence between EpCAM expression in the primary tumor tissue of mBC patients and the CTC detection rates in the corresponding patients is analyzed. Results. (i) MACS recovered significantly more EpCAM (+) than EpCAM (−) tumor cells (p < 0.001) in spiked blood samples. With OQ+ no significantly different recovery rates between EpCAM (+) and EpCAM (−) tumor cells (p = 0.796) were detected. (ii) In mBC patients MACS yielded a significantly higher (p = 0.024) detection rate of EpCAM (+) CTCs. No statistically significant difference (p = 0.070) was found concerning the EpCAM status-based detection rate of CTCs by OQ+. (iii) CTC detection rates are independent of the primary tumors’ EpCAM expression. Conclusions. EpCAM (−) CTCs can not be detected by immunomagnetic EpCAM dependent enrichment methods. EpCAM independent enrichment technologies seem to be superior to detect the entire CTC population. Evaluation of CTCs as prognostic marker should compromise EpCAM (+) and (−) subpopulations

Positive staining for FcεRI α-chain and defensin-5 is observed in serial sections from intestinal tissue.

<p>(A) FcεRI α-chain is detected on the membrane, as well as in the cytoplasm of epithelial cells in small intestine of cancer patient No. 16. FcεRI α-chain positive cells are also found in (B) the colon and (C) a tumor sample from the same patient. (D) Staining with anti-defensin-5 antibodies confirmed that FcεRIα expressing cells at the basis of the small intestinal crypts are Paneth cells. Defensin-5 is expressed also in (E) colon and (F) tumor sample in same areas, but to a lesser extend when compared with FcεRI α-chain staining. Similar staining pattern are observed also for the CD patient No. 8, as FcεRI α-chain positive cells are detected in (G) the epithelium of small intestinal tissue, (H) colon and (I) lesional region. Defensin-5 positive cells are located at (J) the crypt basis of small intestine, (K) along the colon crypt, as well as in (L) the lesional region. Original magnification ×10, inset ×40.</p

Abundant surface and cytoplasmatic FcεRI α-chain expression only in subconfluent human intestinal tumor cell lines.

<p>Immunofluoresence staining for (A–H) FcεRIα is performed in (A, B) subconfluent and (C, D) confluent Caco2/TC7 as well as in (E, F) subconfluent and (G, H) confluent HCT-8. Triton-X-100 permeabilized (A, C, E, G) and untreated cells (B, D, F, H) were compared. (I–L) Representative control staining in subconfluent Caco2/TC7 with the (I, J) anti-lamin A/C or (K, L) unspecific murine IgG2b, (I, K) permeabilized or (J, L) untreated. The blue fluorescence DAPI staining indicates the nuclei. Original magnification ×40.</p

Abundant FcεRI γ-chain expression and IgE binding in subconfluent human intestinal tumor cell lines.

<p>Immunofluorescence staining of (A, D, G, J) FcεRI β- and (B, E, H, K) FcεRI γ-chain are performed in (A–C) subconfluent and (D–F) confluent Caco2/TC7 and in (G–I) subconfluent and (J–L) confluent HCT-8. (C, F, I, L) According to the expression pattern of FcεRI in the subconfluent cells, IgE binding is observed exclusively in subconfluent, non-mature intestinal cells. The blue fluorescence DAPI staining indicates the nuclei. Original magnification ×40.</p