1. Constitutive and regulated secretion of secretory leukocyte proteinase inhibitor by human intestinal epithelial cells 
Mustapha Si-Tahar, Didier Merlin, Shanthi Sitaraman, James L. Madara 
Gastroenterology 
Volume 118, Issue 6, Pages (June 2000)
DOI: /S (00)
Copyright © 2000 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Constitutive expression of SLPI mRNA in 3 human intestinal epithelial cell lines. (A) A map of the human SLPI mRNA fragment for SLPI.25 Base pairs are numbered from the ATG initiation codon. The short black lines represent oligonucleotides used for RT-PCR (see details in Materials and Methods). The arrow indicates the size of the SLPI precursor. (B) A representative result of RT-PCR assay to detect SLPI in the 3 intestinal epithelial cell lines Caco2-BBE, HT29 Cl.19A, and T84. mRNA was extracted from these cells and cDNA was prepared as described in Materials and Methods. PCR products were run on 1% agarose gel and visualized with ethidium bromide. (C) Nucleotide sequence of the enterocyte SLPI cDNA (also available from Genbank under accession no. AF114471).
Gastroenterology  , DOI: ( /S (00) )
Copyright © 2000 American Gastroenterological Association Terms and Conditions

3. Fig. 2
Vectoral SLPI secretion by intestinal epithelial cell lines. Monolayers of Caco2-BBE, HT29 Cl.19A, and T84 cells were grown at 37°C to confluency on microporous membranes. Formation of tight junctions was confirmed by the significant resistance level of the monolayers. Cells were incubated for 5 hours in a serum-free medium, after which the medium was separately removed from the apical (mucosal) and basolateral (serosal) compartments. After a concentration process of the samples, the SLPI concentration was determined by ELISA and expressed as picograms per milliliter. Numbers in brackets refer to the percentage of secretion in the mucosal compartment relative to the total amount of SLPI secreted. Results are mean ± SEM of at least 4 distinct experiments.
Gastroenterology  , DOI: ( /S (00) )
Copyright © 2000 American Gastroenterological Association Terms and Conditions

4. Fig. 3
Regulated secretion of SLPI by cytokines in the intestinal epithelial cell line Caco2-BBE. Monolayers of Caco2-BBE cells were grown at 37°C to confluency on microporous membranes and then incubated for 5 hours in the presence or absence of S. typhimurium (≈5 × 108 bacteria in 25 μL), IFN-γ (1000 U/mL), IL-1β (20 ng/mL), or TNF-α (30 ng/mL). Collected media from each compartment were eventually treated as described in Figure 2. Note that in contrast to cytokines added in both compartments of the cell system, S. typhimurium was only incubated with the apical surface of the monolayers. Likewise, IFN-γ treatment involved a 24-hour preincubation period of this cytokine with the Caco2-BBE monolayers. Results are mean ± SEM of at least 3 distinct experiments.
Gastroenterology  , DOI: ( /S (00) )
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5. Fig. 4
Evidence for the involvement of protein kinase C signaling pathway in SLPI secretion in the intestinal epithelial cell line Caco2-BBE. Monolayers of Caco2-BBE cells were grown at 37°C to confluency on microporous membranes. (A) Cells were incubated for 5 hours in the presence of forskolin (10 μmol/L), thapsigargin (10 μmol/L), or PMA (1.5 μmol/L) or vehicle. Collected media from each compartment were eventually treated as described in Figure 2. (B) Cells were incubated for increasing periods of time in the presence of PMA (1.5 μmol/L) or vehicle. Results are mean ± SEM of at least 4 experiments.
Gastroenterology  , DOI: ( /S (00) )
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6. Fig. 5
SLPI does not modulate barrier function or chloride secretion of the intestinal epithelial cell line T84. Monolayers of T84 cells were grown at 37°C to confluency on microporous membranes and were incubated with recombinant SLPI (5 μmol/L; 60 μg/mL) or vehicle (Veh.). Time course of conductance through the monolayers (i.e., 1/ resistance; A) and Isc (B) were then measured for 2 hours. Also, a modulatory effect of SLPI on agonist-induced chloride secretion was examined by preincubating SLPI for 10 minutes before the addition of 10 μmol/L forskolin (FSK; C). Open arrows indicate the time of addition of SLPI or vehicle; the closed arrow indicates the time of stimulation of epithelial cells by forskolin. Results are mean ± SEM of 3 monolayers for each condition. ●, With SLPI; ○, without SLPI.
Gastroenterology  , DOI: ( /S (00) )
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7. Fig. 6
SLPI provides an antiserine proteinase defense of intestinal epithelial surface. Monolayers of T84 cells were grown at 37°C to confluency on microporous membranes and were incubated with 2.5 μmol/L (A) trypsin or (B) neutrophil elastase. Protective effect of recombinant SLPI was assessed by adding this molecule (1, 5, or 10 μmol/L) 1 minute before the addition of trypsin or elastase. Time course of the conductance (i.e., 1/resistance) through the monolayer was measured for 2 hours. Open arrows indicate the time of addition of SLPI or vehicle; closed arrows indicate the time of addition of trypsin or elastase. Results are mean ± SEM of 3 monolayers for each condition.
Gastroenterology  , DOI: ( /S (00) )
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8. Fig. 7
Antibacterial activity of SLPI against enteropathogenic bacteria. Log-phase S. typhimurium were prepared as described in detail in Materials and Methods and were incubated for 2 hours at 37°C with the indicated concentration of recombinant SLPI. Bacteria suspension were then plated on LB agar medium for 24 hours at 37°C to further analyze the number of CFU. Results are expressed as the percent of control, untreated, bacteria and are the mean ± SEM of 3 distinct experiments.
Gastroenterology  , DOI: ( /S (00) )
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9. Fig. 8
SLPI expression in human intestine. (A) RT-PCR assay to detect SLPI in human colon and jejunum tissues. RNA was isolated from these specimen and cDNA was prepared as described in Materials and Methods. PCR products were run on a 1% agarose gel and visualized with ethidium bromide. (B) Amount of SLPI is expressed in picograms per milligram of total protein, recovered from intestinal lavage fluids of noninflammatory adult patients. Each result represents one individual evaluated in duplicate.
Gastroenterology  , DOI: ( /S (00) )
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