Enterocolitis induced by autoimmune targeting of enteric glial cells: a possible mechanism in Crohn's disease?

Abstract

Early pathological manifestations of Crohn's disease (CD) include vascular disruption, T cell infiltration of nerve plexi, neuronal degeneration, and induction of T helper 1 cytokine responses. This study demonstrates that disruption of the enteric glial cell network in CD patients represents another early pathological feature that may be modeled after CD8(+) T cell-mediated autoimmune targeting of enteric glia in double transgenic mice. Mice expressing a viral neoself antigen in astrocytes and enteric glia were crossed with specific T cell receptor transgenic mice, resulting in apoptotic depletion of enteric glia to levels comparable in CD patients. Intestinal and mesenteric T cell infiltration, vasculitis, T helper 1 cytokine production, and fulminant bowel inflammation were characteristic hallmarks of disease progression. Immune-mediated damage to enteric glia therefore may participate in the initiation and/or the progression of human inflammatory bowel disease.

Figure 1

GFAP-HA transgenic mice: transgene construct and expression. (a) DNA fragment containing the 1.7-kb HA coding sequence placed downstream from the Gfap promoter and upstream of a 0.5-kb segment of the mouse protamine-1 gene (mP1), which provides RNA splicing (thin line) and polyadenylation signals. (b) Southern blot analysis of BamHI-digested genomic DNA from nontransgenic (lane 1) and transgenic (lane 2) progeny of founder GFAP-HA 21. Lanes 3–5: DNA from a nontransgenic mouse supplemented with 1, 10, and 100 copies of transgene per genome, respectively. (c) Northern blot analysis of 60 μg total RNA from brain (lane 1), spinal cord (lane 2), thymus (lane 3) from a transgenic mouse and brain from a nontransgenic littermate (lane 4) using either HA (Upper) or β-actin (Lower) probes. (d) HA (Upper) and β-actin (Lower) mRNA expression assessed by PCR amplification of cDNA from thymus (lane 1), heart (lane 2), brain (lane 3), spinal cord (lane 4), and small intestine from transgenic (lanes 6 and 8) and nontransgenic (lanes 5 and 7) mice. Fragments of 710 and 540 bp were expected for HA and β-actin, respectively. (e) Immunocytochemical detection of HA protein expression on frozen sections: (Upper Left) GFAP-HA transgenic animal with HA expression in the submucosal and myenteric plexus (×450); (Upper Center) higher magnification (×1,000), HA was expressed mainly in enteric nerve plexi; (Upper Right) control mouse, no HA gut expression (×450); (Lower Left) GFAP-HA transgenic animal, spinal cord, HA expression in astrocytes (×450); (Lower Right) GFAP-HA transgenic animal, high magnification of an HA-expressing astrocyte in the brain (×1,300).

Figure 2

(GFAP-HA × CL4-TCR)F₁ double transgenic (DTg) mice develop a fatal disease. (a) Observed distribution of the four different genotypes in pups from crosses of heterozygous CL4-TCR and GFAP-HA Tg mice at days 1 and 21. The frequency of DTg mice at day 21 was significantly less than the expected 25% (P < 0.005). (b) Survival of subgroups of (GFAP-HA × CL4-TCR)F₁ DTg, nontransgenic (NTg), and single CL4-TCR or GFAP-HA Tg littermates. (c) Survival of (GFAP-HA × CL4-TCR)F₁ DTg mice treated with a rat anti-CD8 antibody (n = 10) or control rat IgG (n = 10). (d) Phenotypical appearance of (GFAP-HA × CL4-TCR)F₁ DTg (166), NTg (169), and GFAP-HA Tg (165) littermates at day 5 of postnatal life. (e) Macroscopic appearance of the gastrointestinal tract of a (GFAP-HA × CL4-TCR)F₁ DTg mouse (Upper) and a NTg littermate (Lower) at day 5.

Figure 3

Jejuno-ileo-colitis develops in (GFAP-HA × CL4-TCR)F₁ double transgenic mice. (a) Hematoxylin/eosin-stained section showing normal jejunal histology in a 7-day-old GFAP-HA transgenic mouse. Late-stage lesion of the jejunum (b) and colon (c) in double transgenic mice with mucosal and submucosal edema, epithelial damage, mucosal inflammation, hemorrhage, and necrosis that severely perturbed the crypt-villous architecture and mucosal integrity (×40 for a–c). (d) Normal jejunal histology in GFAP-HA trangenic mice. (e and f) Pathology in double transgenic mice was initially observed in submucosal capillaries and blood vessels (arrows) (×100 for d–f); asterisk in f indicates smooth muscle thickening; (g) with vasodilation, erythrostasis, endothelial hypertrophy and hyperplasia (arrows), and neutrophil (arrowheads) and lymphocyte infiltration. (h) PGP9.5 immunostaining in the jejunal myenteric plexus in GFAP-HA transgenic and (i) double transgenic mice (×400 for g–i).

Figure 4

Small intestine pathology in (GFAP-HA × CL4-TCR)F₁ double transgenic mice. (a) Five-day-old double transgenic pup with moderate inflammation in the gut wall, associated with edema and vasodilatation. Inflammation (anti-CD3) was also observed in the surrounding mesentery. (b) Nontransgenic littermate; normal gut architecture; T cells are concentrated mainly in lymph follicles (×90 for a and b). (c and d) Double transgenic animal, infiltration of the myenteric plexus with T cells (c, ×360; d, ×720); double staining with anti-CD3 (brown, arrows) and GFAP (red, arrowheads). (e) Double transgenic animal; immunohistochemistry with anti-CD8; infiltration of the muscular wall and the myenteric plexus by CD8⁺ T cells (×360). (f) Double transgenic animal; GFAP⁺ immunohistochemistry; edema and inflammation in the mucosa and submucosa; only a few GFAP⁺ cells remain in the myenteric plexus (×450). (g) Double transgenic animal; apoptotic GFAP⁺ cell in the submucosal plexus (×1,300). (h) Double transgenic animal; apoptotic GFAP⁺ cell in the myenteric plexus (×1,300). (i) GFAP⁺ enteric glial cells in the myenteric and submucosal plexi in a normal, nontransgenic animal (×1,000).

Figure 5

GFAP⁺ enteric glial cell populations in human IBD. (A–C) Immunohistochemistry for GFAP in control, involved CD and UC samples, respectively (×100). (D) Box plot demonstrating tissue GFAP levels (ng/mg protein) in clinical biopsies measured by Western blot and ELISA. Clinical groups have been nominated as histological normal controls (CONT; n = 33), noninvolved CD (CD-N; n = 39), involved CD (CD-I; n = 23), noninvolved UC (UC-N; n = 10), and involved UC (UC-I; n = 15). Ileal and colonic biopsies have been pooled because no significant differences were observed between the sites for the different groups. Significant differences (Mann–Whitney U test) from CONT are indicated as (a) included CONT vs. CD-N (P = 0.0005 and P < 0.0001 for Western blot and ELISA, respectively), and CONT vs. UC-I (P = 0.0045 and P = 0.0064 for Western blot and ELISA, respectively). b indicates a significant difference between CD-N and CD-I (P = 0.0058 and P = 0.0001 for Western blot and ELISA, respectively). c indicates a significant difference between UC-N and UC-I (P = 0.023 and P = 0.0009 for Western blot and ELISA, respectively). Significant differences also were recorded between CD-N and UC-N (P = 0.024 and P = 0.0009 for Western blot and ELISA, respectively) and CD-I and UC-I (P = 0.009 and P = 0.0176 for Western blot and ELISA, respectively).