Volume 115, Issue 6, Pages 1426-1438 (December 1998) Differential expression of interleukin 1 receptor antagonist isoforms in human intestinal epithelial cells  Ulrich Böcker, Aderson Damião, Lisa Holt, Dong Soo Han, Christian Jobin, Asit Panja, Lloyd Mayer, R.Balfour Sartor  Gastroenterology  Volume 115, Issue 6, Pages 1426-1438 (December 1998) DOI: 10.1016/S0016-5085(98)70021-6 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Identification of IL-1Ra isoform protein expression in IEC lysates. (A) Only the 18-kilodalton intracellular (ic) IL-1Ra I is found in primary IECs, as judged by the migration patterns of recombinant intracellular IL-1Ra I and II expressed in Caco-2 cells by transient transfection (upper panel, anti-IL-1Ra antibody; lower panel, isotype control). (B) Similarly, the IL-1Ra band of native IECs comigrated with recombinant human intracellular IL-1Ra I and intracellular IL-1Ra I in ovarian EC rather than 17-kilodalton (kDa) recombinant human (rh) secretory IL-1Ra. Concentrated supernatant of IECs cultured for 24 hours did not contain either isoform. (C) Up-regulation of intracellular IL-1Ra I expression was found in IECs isolated from the inactive (I) and actively (A) inflamed areas of the colon or small bowel (SB) of patients with CD or UC. Similar results were obtained for a total of 6 patients with CD and 5 patients with UC. Gastroenterology 1998 115, 1426-1438DOI: (10.1016/S0016-5085(98)70021-6) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Reverse-transcription PCR for IL-1Ra mRNA isoform expression in IECs from normal control patients and patients with CD or UC. A standard curve with secretory (s) IL-1Ra, intracellular (ic) IL-1Ra I, and intracellular IL-1Ra II cDNA was coamplified with cDNA reverse transcribed from total specimen RNA. Band intensity was assessed by densitometry. (A) Result for plasmid cDNA and 1 patient of each group, normal control, and active (A) and inactive (IA) CD and UC. (B) Isoform-specific IL-1Ra mRNA was not detectable in intestinal epithelial lymphocytes and lamina propria lymphocytes. (C and D) Absolute steady-state intracellular IL-1Ra I and actin mRNA levels in isolated IECs by quantitative PCR. P < 0.01 vs. normal. (E) Intracellular IL-1Ra I levels normalized for β-actin mRNA (means ± SEM). *P < 0.05 vs. normal controls. #P < 0.01 vs. normal controls. Gastroenterology 1998 115, 1426-1438DOI: (10.1016/S0016-5085(98)70021-6) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Reverse-transcription PCR for IL-1Ra mRNA isoform expression in IECs from normal control patients and patients with CD or UC. A standard curve with secretory (s) IL-1Ra, intracellular (ic) IL-1Ra I, and intracellular IL-1Ra II cDNA was coamplified with cDNA reverse transcribed from total specimen RNA. Band intensity was assessed by densitometry. (A) Result for plasmid cDNA and 1 patient of each group, normal control, and active (A) and inactive (IA) CD and UC. (B) Isoform-specific IL-1Ra mRNA was not detectable in intestinal epithelial lymphocytes and lamina propria lymphocytes. (C and D) Absolute steady-state intracellular IL-1Ra I and actin mRNA levels in isolated IECs by quantitative PCR. P < 0.01 vs. normal. (E) Intracellular IL-1Ra I levels normalized for β-actin mRNA (means ± SEM). *P < 0.05 vs. normal controls. #P < 0.01 vs. normal controls. Gastroenterology 1998 115, 1426-1438DOI: (10.1016/S0016-5085(98)70021-6) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Immunohistochemical detection of IL-1Ra protein in IECs. Sections are from formalin-fixed, paraffin-embedded mucosal biopsy specimens. (A) Normal control patient with IL-1Ra immunostaining confined to the surface epithelial cells. (B) Active CD with staining of cells extending from the surface to the crypt. (C) Negative control with the same tissue as B, and the primary antibody was preblocked with 10-fold molar excess of recombinant human IL-1Ra (original magnification 100×). Gastroenterology 1998 115, 1426-1438DOI: (10.1016/S0016-5085(98)70021-6) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Expression pattern of intracellular (ic) IL-1Ra I protein and mRNA along the surface-crypt axis. IECs were collected in 6 consecutive fractions collected after 30-minute incubation with dispase with each fraction. (A) Constitutive expression of intracellular IL-1Ra I protein in IECs from a normal control patient, decreasing from surface to crypt. There is a high expression in the crypt with inflammation in IEC fraction VI of the inactive (I) and active (A) areas from a patient with CD. Shown are 1 normal control of 5 controls and 1 patient with IBD of 3 patients with similar results. (B) Constitutive expression of intracellular IL-1Ra I mRNA in IECs from the normal colon and small bowel. rh, recombinant human. Gastroenterology 1998 115, 1426-1438DOI: (10.1016/S0016-5085(98)70021-6) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Expression of IL-1Ra isoform mRNA in parental HT-29/p cells. (A) Quantitation of constitutive secretory (s) IL-1Ra and intracellular (ic) IL-1Ra I and II transcript levels by PCR normalized for actin. (B) *P < 0.0001 vs. intracellular IL-1Ra I/actin Northern blot analysis for intracellular IL-1Ra. (C) Reverse-transcription PCR for secretory IL-1Ra in cells stimulated by IL-1β (1 ng/mL) or PMA (100 ng/mL) for indicated time intervals. Two other experiments gave similar results. Gastroenterology 1998 115, 1426-1438DOI: (10.1016/S0016-5085(98)70021-6) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Expression of IL-1Ra isoform mRNA in parental HT-29/p cells. (A) Quantitation of constitutive secretory (s) IL-1Ra and intracellular (ic) IL-1Ra I and II transcript levels by PCR normalized for actin. (B) *P < 0.0001 vs. intracellular IL-1Ra I/actin Northern blot analysis for intracellular IL-1Ra. (C) Reverse-transcription PCR for secretory IL-1Ra in cells stimulated by IL-1β (1 ng/mL) or PMA (100 ng/mL) for indicated time intervals. Two other experiments gave similar results. Gastroenterology 1998 115, 1426-1438DOI: (10.1016/S0016-5085(98)70021-6) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Expression of intracellular IL-1Ra I protein in parental (undifferentiated) HT-29/p and methotrexate-differentiated HT-29/MTX10-3 cell lines. (A) Western blot analysis with detection of the constitutive 18-kilodalton intracellular IL-1Ra I, but absence of secretory IL-1Ra and intracellular IL-1Ra II. (B) Cellular IL-1Ra content is 1.5-fold higher in HT-29/MTX10-3 by enzyme-linked immunosorbent assay normalized by cell number. (C) IL-1Ra concentration, normalized for total cellular protein, maintaining the 1.5-fold difference between the 2 cell lines (n = 3; means ± SEM; *P < 0.01). Gastroenterology 1998 115, 1426-1438DOI: (10.1016/S0016-5085(98)70021-6) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Expression of intracellular IL-1Ra I protein in parental (undifferentiated) HT-29/p and methotrexate-differentiated HT-29/MTX10-3 cell lines. (A) Western blot analysis with detection of the constitutive 18-kilodalton intracellular IL-1Ra I, but absence of secretory IL-1Ra and intracellular IL-1Ra II. (B) Cellular IL-1Ra content is 1.5-fold higher in HT-29/MTX10-3 by enzyme-linked immunosorbent assay normalized by cell number. (C) IL-1Ra concentration, normalized for total cellular protein, maintaining the 1.5-fold difference between the 2 cell lines (n = 3; means ± SEM; *P < 0.01). Gastroenterology 1998 115, 1426-1438DOI: (10.1016/S0016-5085(98)70021-6) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 (A) Half-life of intracellular (ic) IL-1Ra I mRNA induced by PMA in parental HT-29 (♦) cells and in HT-29 cells permanently differentiated by methotrexate (▩). Cells were stimulated with 100 ng/mL PMA for 1 hour and then treated with 5 μg/mL actinomycin D for 1–5 hours. Numbers under lanes indicate time (h) after addition. (B) mRNA levels were quantitated by exposure to a phosphorimager screen. A second experiment gave a similar result. Gastroenterology 1998 115, 1426-1438DOI: (10.1016/S0016-5085(98)70021-6) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 (A) Half-life of intracellular (ic) IL-1Ra I mRNA induced by PMA in parental HT-29 (♦) cells and in HT-29 cells permanently differentiated by methotrexate (▩). Cells were stimulated with 100 ng/mL PMA for 1 hour and then treated with 5 μg/mL actinomycin D for 1–5 hours. Numbers under lanes indicate time (h) after addition. (B) mRNA levels were quantitated by exposure to a phosphorimager screen. A second experiment gave a similar result. Gastroenterology 1998 115, 1426-1438DOI: (10.1016/S0016-5085(98)70021-6) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 8 (A) Caco-2 cells were permanently transfected with an expression vector for intracellular (ic) IL-1Ra I (▩). An empty vector was used as a control (♦). IL-8 secretion was measured over a period of 48 hours after stimulation by IL-1β (1 ng/mL). Mean values of triplicate experiments. Two more experiments on different clones gave similar results. (B) Inhibition of IL-1β–stimulated IL-8 secretion in nontransfected Caco-2 cells by exogenous intracellular IL-1Ra I. Mean values of triplicate experiments. Two more experiments gave similar results. Gastroenterology 1998 115, 1426-1438DOI: (10.1016/S0016-5085(98)70021-6) Copyright © 1998 American Gastroenterological Association Terms and Conditions