Monocyte/macrophage activation by normal bacteria and bacterial products: implications for altered epithelial function in Crohn's disease

Abstract

Intestinal immune cells are less reactive than those in the peripheral blood; however, such cells from patients with Crohn's disease may be more responsive to bacterial products. Our study examined if nonpathogenic bacteria or lipopolysaccharide (LPS), can affect epithelial function in the presence of monocytes/macrophages. Lamina propria mononuclear cells (LPMCs) and peripheral blood monocytes (PBMs) were obtained from patients with Crohn's disease and control patients. Filter-grown T84 epithelial monolayers were co-cultured with nonactivated or LPS-activated LPMCs or PBMs for 48 hours. Epithelial secretory [baseline short-circuit current (Isc) and DeltaIsc to forskolin] and barrier (transepithelial electrical resistance) parameters were measured in Ussing chambers. LPS-activated PBMs from both controls and patients with Crohn's disease significantly increased Isc ( approximately 300%) and reduced transepithelial electrical resistance ( approximately 40%). Epithelial function was not altered after co-culture with control LPMCs +/- LPS. However, LPMCs from patients with Crohn's disease spontaneously secreted tumor necrosis factor-alpha, and induced epithelial changes similar to those produced by LPS-activated PBMs. Co-culture with control Escherichia coli and PBMs induced comparable changes in epithelial physiology, which were abrogated by anti-tumor necrosis factor-alpha antibody. We conclude that LPMCs of patients with Crohn's disease are spontaneously activated, possibly by gram-negative luminal bacteria, and can directly cause significant alterations in epithelial ion transport and barrier functions.

Figure 1.

Percent changes from control values (T84 cells alone) in epithelial baseline Isc (A), ΔIsc in response to forskolin (FSK) (B), and transepithelial resistance (C) of T84 monolayers after 48 hours of co-culture with normal or Crohn’s disease (CD) PBMs in the presence or absence of LPS (n = 12 to 14 donors) [mean ± SEM, two to four monolayers per condition; *, P < 0.05 and **, P < 0.005 compared to control (100%)].

Figure 2.

Basal (−LPS) or stimulated (+LPS) TNF-α production by PBMs (A) and LPMCs (B) from normals or patients with Crohn’s disease (CD) after 24 hours of co-culture (n = 6 to 8 donors). Each symbol represents an individual cell donor.

Figure 3.

Percent changes from control values (T84 cells alone) in epithelial baseline Isc (A), ΔIsc in response to forskolin (FSK) (B), and transepithelial resistance (C) of T84 monolayers after 48 hours of co-culture with normal or Crohn’s disease LPMCs in the presence or absence of LPS (n = 8 to 10 specimens per condition). Data for small bowel (SB) and large bowel (LB) are shown separately and combined as Crohn’s total; statistical analyses performed on grouped Crohn’s total data only [mean ± SEM, two to four monolayers per condition; *, P < 0.05 and **, P <0.005 compared to control (100%); control values: baseline Isc range = 0 to 6 (mean, 2.4) μA/cm²; ΔIsc to FSK range = 30 to 154 (mean, 70) μA/cm²; TER range = 770 to 3,333 (mean, 1,784) Ω/cm²].

Figure 4.

Percent changes from control values (T84 cells alone) in epithelial secretory responses; baseline Isc (A) and ΔIsc in response to forskolin (FSK) (B) of T84 monolayers after 48 hours of co-culture with control E. coli alone or E. coli and PBMs in the presence or absence of anti-TNF-α antibody (aTNF-α) (n = 6 to 8 donors) [mean ± SEM, two to four monolayers per condition; *, P < 0.05 compared to control (100%); control values: baseline Isc range = 1 to 3 (mean, 1.4) μA/cm²; ΔIsc to FSK range = 66 to 118 (mean, 92) μA/cm²].

Figure 5.

Changes from control values (T84 cells alone) in epithelial barrier function measured by transepithelial resistance (A) and mucosal-to-serosal flux of ⁵¹Cr-EDTA across T84 monolayers (B) after 48 hours of co-culture with control E. coli alone or E. coli and PBMs in the presence or absence of anti-TNF-α antibody (aTNF-α) (n = 6 to 8 donors) [mean ± SEM, two to four monolayers per condition; *, P < 0.05 compared to control (100%); control TER range = 860 to 1,620 (mean, 1,093) Ω/cm²].