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. 1998 Apr;42(4):538–545. doi: 10.1136/gut.42.4.538

Protein transport and processing by human HT29-19A intestinal cells: effect of interferon γ

K Terpend 1, F Boisgerault 1, M Blaton 1, J Desjeux 1, M Heyman 1
PMCID: PMC1727077  PMID: 9616317

Abstract

Background—The nature of the breakdown products produced in enterocytes during epithelial transport of intact proteins may be critical in determining the functional consequences of protein absorption. 
Aim—(a) To measure the transepithelial transport of horseradish peroxidase (HRP) and to identify the nature of HRP breakdown products released on the basal side of enterocytes and (b) to assess the role of interferon γ (IFNγ) on HRP transport and processing. 
Methods—HT29-19A intestinal cells were used to assess transepithelial transport of HRP in Ussing chambers, and the nature of breakdown products in the basal compartment was analysed by high performance liquid chromatography (HPLC). 
Results—(1) In control conditions, [3H]HRP equivalent fluxes (3135 (219) ng/h per cm2; mean (SEM)) comprised 50% amino acids, 40% peptides, and 10% intact HRP. Steric exclusion HPLC of the breakdown products indicated a wide range of molecular masses including a major peptide of about 1150 Da. Lysosomal aspartyl and thiol proteases were expressed but no HLA-DR surface expression was noted. (2) At 48 to 72 hours after IFNγ stimulation, [3H]HRP equivalent fluxes increased significantly (7392 (1433) ng/h per cm2) without modification of the relative proportions of amino acids, peptides, and intact HRP, and without modification of the distribution of breakdown products in HPLC. Lysosomal protease activities were not modified by IFNγ but HLA-DR expression was increased. 
Conclusion—Intestinal cells are able to process HRP into peptides potentially capable of stimulating the immune system. IFNγ stimulates the transport and processing of HRP thus increasing the antigenic load in the intestinal mucosa. 



Keywords: enterocyte; transcytosis; macromolecular degradation; HPLC; mucosal immunity

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Figure 1 .

Figure 1

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Effect on paracellular transport of placing interferon γ (IFNγ) on the basal or apical side of HT29-19A intestinal cell monolayers for 48 or 72 hours. Paracellular permeability was measured as (A) ionic conductance (G), and (B) mannitol and (C) Na fluxes. *Significantly different from untreated cells (p⩽0.02); †significantly different from time matched cells after IFNγ treatment on the apical side (p⩽0.03). n = 8 to 20 filter grown intestinal monolayers. 


Figure 2 .

Figure 2

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Effect of placing interferon γ (IFNγ) on the basal or apical side of HT29-19A intestinal cell monolayers for 48 or 72 hours, on the transepithelial transport of (A) 3H labelled horseradish peroxidase ([3H]HRP) in (B) intact or (C) degraded form. *Significantly different from untreated cells (p⩽0.008); †significantly different from time matched apical conditions (p⩽0.01). n = 8 to 17 filter grown monolayers. 


Figure 3 .

Figure 3

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Processing of 3H labelled horseradish peroxidase ([3H]HRP) during transport. Incubation for 48 hours with interferon γ (IFNγ) increased 3H equivalent HRP transport (total HRP) without significantly modifying the percentages of amino acids, peptides, and intact HRP. n = 5 to 6 separate experiments. 


Figure 4 .

Figure 4

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3H labelled metabolites formed after transepithelial transport of 3H labelled horseradish peroxidase ([ 3H]HRP) across intestinal HT29-19A cells, as shown by reverse phase high performance liquid chromatography (RP-HPLC). [3H]HRP before transport (A), and 3H labelled metabolites and [3H]HRP after transport in untreated cells (B) and in cells treated with interferon γ (IFNγ) for 48 hours (C). This elution profile is representative of two separate experiments. 


Figure 5 .

Figure 5

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3H labelled peptides formed during transepithelial transport of 3H labelled horseradish peroxidase ([ 3H]HRP) across intestinal HT29-19A cells as shown by steric exclusion HPLC. [3H]HRP before transport (A),[3H]HRP after 4.5 hours in the apical compartment (B),[3H]metabolites after transepithelial transport in the basal compartment, in untreated cells (C) and cells treated with interferon γ (IFNγ) (D). This chromatogram is representative of two separate experiments. 


Figure 6 .

Figure 6

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HLA-DR molecule expression on the surface of untreated intestinal HT29-19A cells, and cells treated on their basal side with 100 U/ml recombinant human interferon γ (IFNγ) for 48 or 72 hours. A monoclonal antibody recognising HLA-DR (clone L243) was used. Untreated cells did not express HLA-DR. After 48 hours of IFNγ treatment, HLA-DR expression was significant, and after 72 hours, it had greatly increased. A positive control was run with EBV-B cells. 


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