Coexpression and interaction of CXCL10 and CD26 in mesenchymal cells by synergising inflammatory cytokines: CXCL8 and CXCL10 are discriminative markers for autoimmune arthropathies

Abstract

Leukocyte infiltration during acute and chronic inflammation is regulated by exogenous and endogenous factors, including cytokines, chemokines and proteases. Stimulation of fibroblasts and human microvascular endothelial cells with the inflammatory cytokines interleukin-1beta (IL-1beta) or tumour necrosis factor alpha (TNF-alpha) combined with either interferon-alpha (IFN-alpha), IFN-beta or IFN-gamma resulted in a synergistic induction of the CXC chemokine CXCL10, but not of the neutrophil chemoattractant CXCL8. In contrast, simultaneous stimulation with different IFN types did not result in a synergistic CXCL10 protein induction. Purification of natural CXCL10 from the conditioned medium of fibroblasts led to the isolation of CD26/dipeptidyl peptidase IV-processed CXCL10 missing two NH2-terminal residues. In contrast to intact CXCL10, NH2-terminally truncated CXCL10(3-77) did not induce extracellular signal-regulated kinase 1/2 or Akt/protein kinase B phosphorylation in CXC chemokine receptor 3-transfected cells. Together with the expression of CXCL10, the expression of membrane-bound CD26/dipeptidyl peptidase IV was also upregulated in fibroblasts by IFN-gamma, by IFN-gamma plus IL-1beta or by IFN-gamma plus TNF-alpha. This provides a negative feedback for CXCL10-dependent chemotaxis of activated T cells and natural killer cells. Since TNF-alpha and IL-1beta are implicated in arthritis, synovial concentrations of CXCL8 and CXCL10 were compared in patients suffering from crystal arthritis, ankylosing spondylitis, psoriatic arthritis and rheumatoid arthritis. All three groups of autoimmune arthritis patients (ankylosing spondylitis, psoriatic arthritis and rheumatoid arthritis) had significantly increased synovial CXCL10 levels compared with crystal arthritis patients. In contrast, compared with crystal arthritis, only rheumatoid arthritis patients, and not ankylosing spondylitis or psoriatic arthritis patients, had significantly higher synovial CXCL8 concentrations. Synovial concentrations of the neutrophil chemoattractant CXCL8 may therefore be useful to discriminate between autoimmune arthritis types.

Figure 1

CXCL8 and CXCL10 induction in fibroblasts by IL-1β and interferons. Confluent fibroblast monolayers were incubated with IL-1β in combination with IFN-α, IFN-β or IFN-γ. Results represent the mean CXCL8 and CXCL10 protein concentration (ng/ml) measured in the culture supernatant (three or more independent experiments).

Figure 2

CXCL8 and CXCL10 induction in fibroblasts by tumour necrosis factor alpha and interferons. Confluent fibroblast monolayers were incubated with tumour necrosis factor alpha (TNF-α) in combination with IFN-α, IFN-β or IFN-γ. Results represent the mean CXCL8 and CXCL10 concentration (ng/ml) measured in the culture supernatant (three or more independent experiments).

Figure 3

CXCL10 induction by combinations of interferons in fibroblasts and human microvascular endothelial cells. Monolayers of fibroblasts or human microvascular endothelial cells (HMVEC) were incubated with combinations of IFN-α or IFN-β and IFN-γ. Results represent the mean CXCL10 concentration (ng/ml) measured in the culture supernatant (three or more independent experiments).

Figure 4

CXCL8 and CXCL10 induction in human microvascular endothelial cells by IL-1β and interferons. Human microvascular endothelial cells (HMVEC) were incubated with IL-1β in combination with IFN-α, IFN-β or IFN-γ. Results represent the mean CXCL8 and CXCL10 concentration (ng/ml) measured in the culture supernatant (three or more independent experiments).

Figure 5

CXCL8 and CXCL10 induction in HMVEC by tumour necrosis factor alpha and interferons. Human microvascular endothelial cells (HMVEC) were incubated with tumour necrosis factor alpha (TNF-α) in combination with IFN-α, IFN-β or IFN-γ. Results represent the mean CXCL8 and CXCL10 concentration (ng/ml) measured in the culture supernatant (three or more independent experiments).

Figure 6

Reverse-phase HPLC purification of fibroblast-derived CXCL10. Semi-purified fibroblast-derived CXCL10 was subjected to C8 reverse-phase HPLC. Proteins were eluted in an acetonitrile gradient (dashed line) and UV absorbance was detected at 214 nm (solid line). CXCL10 immunoreactivity in the column fractions was detected by ELISA (histograms).

Figure 7

Identification of fibroblast-derived CXCL10. The relative molecular mass (M_r) of reverse-phase-HPLC-purified CXCL10 was determined by electrospray ion trap mass spectrometry. Results show the (a) averaged and (b) averaged deconvoluted spectra of CXCL10 that eluted in between 26% and 28% acetonitrile from the C8 column (Figure 6). The amino acids cleaved off (one-letter code), explaining the differences between the CXCL10 isoforms, are indicated on top of the averaged deconvoluted spectrum. Both NH₂-terminally truncated, COOH-terminally intact CXCL10(3–77), CXCL10(4–77), CXCL10(5–77), CXCL10(6–77), and NH₂-terminally and COOH-terminally cleaved CXCL10(3–73), CXCL10(4–73), CXCL10(5–73) and CXCL10(6–73) were identified. The deviation between the theoretical and the experimentally determined average M_r for each amino acid is indicated below the one-letter code.

Figure 8

CXCR3-dependent signalling. Serum-starved Chinese hamster ovary CXCR3 cells were treated with Ham's F-12 medium supplemented with 0.5% foetal bovine serum (FBS) or stimulated with CXCL10 or NH₂-terminally truncated CXCL10(3–77) at a concentration of 1, 10 or 100 ng/ml (in Ham's F-12 supplemented with 0.5% FBS). The reaction was stopped after 5 minutes and the cells were lysed. The level of extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation or protein kinase B/Akt phosphorylation in the cell lysate was determined with specific ELISAs for phosphoERK or phosphoAkt. The mean values (n = 4) and standard errors are indicated. *Statistically significant differences (Mann–Whitney U test) from control (P < 0.05).

Figure 9

Detection of CD26 by Fluorescence-activated cell sorting (FACS) analysis. Expression of CD26 was detected by FACS analysis. (a) Expression level of CD26 on unstimulated fibroblasts. Background staining with secondary antibody only (black histograms) was compared with specific CD26-staining (open histograms). Control staining with isotype antibodies resulted in a similar histogram as with secondary antibody alone. Confluent fibroblast monolayers were left untreated (Co) or were treated with IL-1β (100 U/ml), tumour necrosis factor alpha (TNF-α) (10 ng/ml), IFN-γ (200 ng/ml) or with combinations of these cytokines. (b) Regulation of CD26 expression as the percentage of the relative mean fluorescence intensity (MFI) for untreated fibroblasts (± standard error of the mean). The mean MFI of four experiments is shown (except for treatment with IFN-γ alone, for which n = 3). Statistical analysis was performed with the Mann–Whitney U test, *P < 0.05.

Figure 10

Detection of dipeptidyl peptidase IV activity. (a) Soluble dipeptidyl peptidase IV (DPP IV) activity in serum-free conditioned medium from fibroblast cultures or (b)–(d) the activity of DPP IV associated with fibroblast membranes was evaluated by detecting the release of p-nitroanilide from GlyPro-p-nitroanilide (increase in UV absorption at 400 nm (OD₄₀₀) compared with culture medium or untreated cells). Fibroblasts were either left untreated or were treated with the cytokines IFN-α (1000 U/ml), IFN-β (1000 U/ml), IFN-γ (200 ng/ml), IL-1β (10 U/ml) or tumour necrosis factor alpha (TNF-α) (10 ng/ml), or combinations thereof. A significantly increased membrane-bound DPP IV activity was detected on fibroblasts treated with IFN-γ (P < 0.0008), with IL-1β + IFN-γ (P < 0.0004) or with TNF-α + IFN-γ (P < 0.002). Statistical analysis for DPP IV activity on cytokine-treated versus untreated fibroblasts (n = 4 for cytokine combinations and n = 8 for individual cytokines) was performed with the Student t test with Bonferroni correction.

Figure 11

CXCL8 and CXCL10 in synovial fluid of arthritis patients. (a) CXCL10 and (b) CXCL8 concentrations were measured by ELISAs in synovial fluids of patients with ankylosing spondylitis (AS), psoriatic arthritis (PsA) and rheumatoid arthritis (RA), and were compared with chemokine concentrations in the metabolic arthritis patients with crystal-induced arthritis (CA). The detection limits of the ELISAs for the synovial concentrations of CXCL8 and CXCL10 are indicated on the y axis (logarithmic scale) and were 0.25 ng/ml and 1 ng/ml, respectively. Statistical analysis was performed with the median levels (dashed bars) using the nonparametric Mann–Whitney U test.