Disordered macrophage cytokine secretion underlies impaired acute inflammation and bacterial clearance in Crohn's disease

Abstract

The cause of Crohn's disease (CD) remains poorly understood. Counterintuitively, these patients possess an impaired acute inflammatory response, which could result in delayed clearance of bacteria penetrating the lining of the bowel and predispose to granuloma formation and chronicity. We tested this hypothesis in human subjects by monitoring responses to killed Escherichia coli injected subcutaneously into the forearm. Accumulation of (111)In-labeled neutrophils at these sites and clearance of (32)P-labeled bacteria from them were markedly impaired in CD. Locally increased blood flow and bacterial clearance were dependent on the numbers of bacteria injected. Secretion of proinflammatory cytokines by CD macrophages was grossly impaired in response to E. coli or specific Toll-like receptor agonists. Despite normal levels and stability of cytokine messenger RNA, intracellular levels of tumor necrosis factor (TNF) were abnormally low in CD macrophages. Coupled with reduced secretion, these findings indicate accelerated intracellular breakdown. Differential transcription profiles identified disease-specific genes, notably including those encoding proteins involved in vesicle trafficking. Intracellular destruction of TNF was decreased by inhibitors of lysosomal function. Together, our findings suggest that in CD macrophages, an abnormal proportion of cytokines are routed to lysosomes and degraded rather than being released through the normal secretory pathway.

Figure 1.

Neutrophil accumulation and subsequent clearance of E. coli from the tissues is markedly delayed in a dose-dependent manner in CD. ¹¹¹Indium-labeled autologous neutrophils were injected intravenously at the same time as killed E. coli were injected subcutaneously into each forearm. (a) Radioactivity measured over the injection sites showed a much smaller proportion of labeled cells accumulating in CD subjects. (b) γ-Camera image of a CD patient at 24 h after injection, demonstrating focal accumulations of radioactivity at bacterial injection sites (arrows) and confirming lack of bowel inflammation. (c) ³²P-labeled killed E. coli were injected into the subcutaneous tissues of the forearm and radioactivity was measured at the skin surface. Clearance of radioactivity was much slower in CD than in HC or UC. Extrapolating these curves indicated that almost complete removal (99%) would take 10.2 and 7.1 d in HC and UC subjects, respectively, compared with 44.3 d in CD. (d and e) Effect of increasing bacterial dose from 10⁵ to 10⁸ on blood flow at injection site (d) and bacterial clearance (e). The numbers of subjects studied in the dose response experiment are depicted in e. All results are expressed as mean ± SEM (**, P < 0.01; ***, P < 0.001).

Figure 2.

Proinflammatory cytokine secretion by macrophages from CD patients is deficient in response to E. coli. Supernatants from macrophages stimulated for 24 h with HkEc were tested for the levels of cytokines and chemokines. (a) Macrophages from HC subjects released varying amounts of cytokines and chemokines after HkEc stimulation. (b) Cytokine and chemokine release expressed as a percentage of that secreted by HC cells (blue bar) from ileal and colonic CD patients. The numbers of subjects in each group are shown on left; each patient was used once. All results are expressed as mean ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 3.

Abnormal transcriptional profiles of CD macrophages on Affymetrix gene arrays. (a) Venn diagram shows the number of probe sets differing in expression (P < 0.01) between ileal and colonic CD versus HC macrophages in the unstimulated state and after exposure to HkEc. (b and c) As in a, but comparing differentially expressed probe sets between ileal and colonic CD and UC versus HC in unstimulated (b) and HkEc-stimulated (c) cells. (d) i-iv designate overlaps of differentially expressed genes of known function after HkEc stimulation between disease groups, as labeled in Venn diagrams.

Figure 4.

Intracellular levels of TNF are lower in CD, despite normal transcription and mRNA stability, but restored to normal in the presence of Bref-A and lysosome inhibitors. (a) Macrophages transfected with an adenoviral vector containing a TNF promoter and luciferase reporter demonstrated equivalent TNF transcription levels in HC (n = 8) and CD (n = 7) subjects. (b) TNF mRNA stability is comparable in HC and CD macrophages after stimulation with HkEc. (c) Intracellular levels of TNF after HkEc stimulation with or without Bref-A were determined by Western blotting. (d) CD contained significantly lower levels of intracellular TNF than HC macrophages after HkEc stimulation but returned to normal levels with the inclusion of Bref-A (n = 6 in all groups). (e) Intracellular levels of TNF after HkEc stimulation with or without lysosomal inhibitors were determined by Western blotting. (f) Inhibitors of lysosomal proteolysis increase intracellular TNF levels in HC and CD macrophages (n = 4 in all groups; gray line denotes level of HkEc alone). Significance levels are compared with HkEc alone. All patients in these studies had colonic CD and subjects were used once per assay. Results are shown as mean ± SEM (*, P < 0.05).

Figure 5.

Intracellular levels of chemokines and cytokines after HkEc stimulation in the presence of vesicle trafficking and lysosomal inhibitors. (a) Intracellular cytokine array profiles obtained from macrophages stimulated with HkEc in the presence of absence of either Bref-A or monensin. (b) Cytokine array map. (c) Various chemokines and cytokines demonstrate reduced intracellular levels in CD macrophages after HkEc stimulation and are either normalized or elevated after Bref-A or monensin treatment (n = 4 in all groups). All patients studied had colonic CD and subjects were used once per assay. Results shown are mean ± SEM (*, P < 0.05; ***, P < 0.001).