Identification of myeloid cells in the human enthesis as the main source of local IL-23 production

Abstract

Objective: We investigated whether the normal human spinal enthesis contained resident myeloid cell populations, capable of producing pivotal proinflammatory cytokines including tumour necrosis factor (TNF) and interleukin (IL)-23 and determined whether these could be modified by PDE4 inhibition.

Methods: Normal human enthesis soft tissue (ST) and adjacent perientheseal bone (PEB) (n=15) were evaluated using immunohistochemistry (IHC), digested for myeloid cell phenotyping, sorted and stimulated with different adjuvants (lipopolysaccharide and mannan). Stimulated enthesis fractions were analysed for inducible production of spondyloarthropathy disease-relevant mediators (IL-23 full protein, TNF, IL-1β and CCL20). Myeloid populations were also compared with matched blood populations for further mRNA analysis and the effect of PDE4 inhibition was assessed.

Results: A myeloid cell population (CD45+ HLADR+ CD14+ CD11c+) phenotype was isolated from both the ST and adjacent PEB and termed 'CD14+ myeloid cells' with tissue localisation confirmed by CD14+ IHC. The CD14- fraction contained a CD123+ HLADR+ CD11c- cell population (plasmacytoid dendritic cells). The CD14+ population was the dominant entheseal producer of IL-23, IL-1β, TNF and CCL20. IL-23 and TNF from the CD14+ population could be downregulated by a PDE4I and other agents (histamine and 8-Bromo-cAMP) which elevate cAMP. Entheseal CD14+ cells had a broadly similar gene expression profile to the corresponding CD14+ population from matched blood but showed significantly lower CCR2 gene expression.

Conclusions: The human enthesis contains a CD14+ myeloid population that produces most of the inducible IL-23, IL-1β, TNF and CCL20. This population has similar gene expression profile to the matched blood CD14+ population.

Keywords: IL-23; enthesis; myeloid cell; pde4 inhibitors; spondyloarthritis.

Figure 1

Immunohistochemistry of CD14+ cells in (A) perientheseal bone (PEB) and (B) entheseal soft tissue (ST). Enthesis samples were separated into PEB and ST and CD14+ and CD14− cells sorted. (C–J) CD14+ and CD14− fractions were stimulated with LPS/interferon gamma (IFNү) or mannan for 48 hours. TNF, IL-23, CCL20 and IL-1β levels were measured by ELISA. n=5. Two-way analysis of variance (ANOVA) with Bonferroni multiple comparisons test was performed. *Significance from relevant unstimulated control, for example, CD14+ or CD14− unstimulated. (K) PEB was stimulated with LPS/IFNү in the presence of Golgi plug for 16 hours. Cells were surface stained for CD14+ and CD14− cells and intracellular IL-23p19 were measured. Blue histograms represent unstimulated and red stimulated. (L) Change in IL-23p19 median fluorescence intensity (MFI), following LPS/IFNү stimulation in both CD14+ and CD14− gates. n=3. Paired t-test. *p<0.05; **p<0.01; ***p<0.001. IL, interleukin; LPS, lipopolysaccharide; PEB, perientheseal bone; ST, soft tissue; TNF-α, tumour necrosis factor alpha.

Figure 2

(A–D) CD14+ fraction cells were isolated from the PEB and ST as before (n=5), and stimulated with LPS/IFNү and mannan as before, with and without PDE4I rolipram, IL-23 and TNF were measured by ELISA. (E, F) Healthy blood CD14+ cells were stimulated with LPS/IFNү (E) or mannan (F) with and without cAMP-elevating agents; rolipram, 8-Br-cAMP and histamine for 48 hours (n=7), IL-23 secretion was measured by ELISA. One-way analysis of variance (ANOVA) with Bonferroni multiple comparisons test was performed. (E, F) Significance from stimulated (LPS or mannan). (G) CD14+ cells were isolated from the PEB or matched blood and transcript analysis was performed (n=4). Black bars represent those higher expressed in blood, white bars those with higher expression in PEB. Genes highlighted in blue were undetectable in both blood and PEB. A paired t-test was performed on ΔCt values for PEB versus blood. *p<0.05; **p<0.01; ***p<0.001. IFN, interferon; IL, interleukin; LPS, lipopolysaccharide; PEB, perientheseal bone; ST, soft tissue; TNF, tumour necrosis factor; VEGF, vascular endothelial growth factor.