Synergistic induction of CXCL10 ligands in fibroblasts and endothelial cells by inflammatory cytokines.
The lymphocyte chemotactic CXCR3 ligands are known to be inducible by IFNs, whereas IL-1β and TNF-α are potent inducers of several other chemokines such as the main CXCR1 and CXCR2 ligand CXCL8. IL-1β, TNF-α and IFNs are often coproduced during inflammation. The ability of combinations of these cytokines to induce CXCL8 and CXCL10 in fibroblasts was therefore investigated.
Diploid fibroblasts were grown to confluency and were stimulated with IL-1β (0.001–10 U/ml) or TNF-α (0.001–10 ng/ml) in conditioned media in the presence of IFN-α (10–10,000 U/ml), IFN-β (10–1000 U/ml) or IFN-γ (2–200 ng/ml) for 72 hours. The culture medium was then analysed for CXCL10 production by specific ELISA. Although IL-1β and TNF-α as well as IFN-α, IFN-β or IFN-γ were rather weak inducers of CXCL10 (1–5 ng/ml) in fibroblasts as single agents, all combinations provided a dose-dependent synergistic induction yielding a 3-fold to 30-fold increase of CXCL10 production (5–150 ng/ml) (Figures and , left panels). In particular, induction of fibroblasts with IL-1β or TNF-α together with IFN-γ (2–200 ng/ml) provided a strong synergistic effect (up to 50-fold increase above the additive effect for IL-1β and IFN-γ). Stimulation of fibroblasts with IFN-α plus IFN-γ or with IFN-β plus IFN-γ (Figure ), however, only yielded a weak synergistic CXCL10 induction and the total CXCL10 production remained low (≤1 ng/ml). This indicates that the synergy with IFN-γ does not indirectly depend on the induction of IFN-β on the fibroblasts by IL-1β or TNF-α.
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 (more ...)
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. (more ...)
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 (more ...)
In addition, the production of CXCL8, the chemokine with the highest specific activity on neutrophilic granulocytes, was determined after stimulation of fibroblasts with IL-1β or TNF-α in the presence of IFN-α, IFN-β or IFN-γ (Figures and , right panels). IL-1β (1 U/ml) and TNF-α (10 ng/ml) alone induced more than 100 ng/ml CXCL8. The presence of IFN-β or IFN-γ rather moderately and dose-dependently inhibited the production of CXCL8 in response to IL-1β. Finally, fibroblast treatment with single or combined IFN types did not result in CXCL8 production (data not shown). It can be concluded that IFNs in fibroblasts inhibit CXCL8 production, whereas IFNs in combination with IL-1β or TNF-α synergistically stimulate production of CXCL10.
HMVEC not only play a crucial role in leukocyte extravasation during inflammatory processes, but also form a rich source of chemokines and are targets for angiogenic chemokines (e.g. CXCL8) and antiangiogenic chemokines (e.g. CXCL10). Similar to fibroblasts, synergistic CXCL10 induction occurred between IL-1β or TNF-α and IFN-γ, whereas the cooperation between IL-1β or TNF-α and IFN-α or IFN-β was less pronounced (IFN-β) to rather weak (IFN-α) (Figures and ). HMVEC, in contrast to fibroblasts, however, required 100-fold lower amounts of IFN-γ to obtain similar levels of CXCL10 in the culture supernatant. Moreover, the cell density of the in vitro cultures was about fivefold lower for HMVEC compared with fibroblasts. As in fibroblasts, no synergy between IL-1β or TNF-α and IFNs was observed for CXCL8 production in HMVEC (Figures and ).
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 (more ...)
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. (more ...)
Biochemical and biological characterisation of CXCL10 isoforms from fibroblasts
The conditioned medium from fibroblast cultures stimulated with inflammatory mediators was first concentrated by adsorption to controlled pore glass, and then chemokine fractionation was achieved upon subsequent heparin Sepharose affinity chromatography. The CXCL10 immunoreactivity eluted in a single peak between 0.7 M and 1.15 M NaCl, after the CXCL8 peak (data not shown). Further purification of CXCL10 was obtained by cation exchange chromatography. CXCL10 eluted between 0.65 M and 0.75 M NaCl from the Mono S column and was finally purified to homogeneity by C8 RP-HPLC (Figure ). The majority of CXCL10 immunoreactivity eluted from the C8 column between 40 minutes and 46 minutes (26–29% acetonitrile).
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 (more ...)
Mass spectrometry revealed that at this stage CXCL10 was still heterogeneous since molecules with different Mr were detected upon deconvolution of the spectra (Figure ). The Mr of all observed proteins, however, fitted with the theoretical Mr of specific NH2-terminally truncated and/or COOH-terminally truncated forms of CXCL10. Edman degradation confirmed the existence of the different NH2-terminally truncated CXCL10 forms.
Figure 7 Identification of fibroblast-derived CXCL10. The relative molecular mass (Mr) 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 (more ...)
Comparison of signalling activity of intact and truncated CXCL10
The two most abundant CXCL10 isoforms were missing two or three NH2
-terminal residues. In particular, the CXCL10(3–73) isoform missing its two NH2
-terminal residues was interesting, since this isoform can be generated in vitro
through proteolytic cleavage of CXCL10 by soluble DPP IV (designated CD26) [22
]. CHO cells transfected with CXCR3 were incubated with different concentrations of recombinant intact and CD26/DPP IV-truncated CXCL10. Intact CXCL10 at a concentration as low as 1 ng/ml was able to induce significant ERK1/2 phosphorylation in CHO/CXCR3 cells within 5 minutes (Figure ). Phosphorylation of Akt was obtained upon stimulation of the CHO/CXCR3 cells with 100 ng/ml intact CXCL10. In contrast, no ERK1/2 or Akt phosphorylation was observed upon treatment of CHO/CXCR3-transfected cells with CXCL10(3–77) at concentrations up to 100 ng/ml.
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 (more ...)
Regulation of CD26/DPP IV expression and DPP IV activity in fibroblasts
The fact that fibroblasts are a cellular source of CXCL10 missing the two NH2
-terminal residues indicates that CD26/DPP IV may be functionally expressed on these cells. In addition to CD26, the related enzyme fibroblast activation protein, is also capable of cleaving post-proline bonds and may be responsible for the observed DPP IV activity [31
]. FACS analysis on fibroblast cultures, used to study CXCL10 expression, confirmed the presence of membrane-bound CD26 protein (Figure ). Since CD26 also exists in a shed soluble form [32
], DPP IV activity was analysed in fibroblast cultures as well as in the culture supernatant with a substrate conversion assay. Although membrane-bound DPP IV activity was detected on fibroblasts, there was no soluble DPP IV activity present in the culture supernatant (Figure ).
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 (more ...)
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 (more ...)
To investigate whether DPP IV activity (or CD26 expression) could be upregulated in fibroblasts by cytokines under similar conditions to those used to induce CXCL10, cell cultures were stimulated with IL-1β, TNF-α, IFN-α, IFN-β or IFN-γ, or mixtures thereof, in serum-free medium. Fibroblast-derived DPP IV activity was, however, not detected in the conditioned medium with the substrate conversion assay (Figure ) and no soluble CD26 protein was detected by ELISA (data not shown), – although CXCL10 immunoreactivity was produced as previously shown (Figure ). Induction of fibroblasts with IL-1β or TNF-α in the presence or absence of IFN-α or IFN-β did not significantly affect membrane-bound activity of DPP IV on fibroblasts (Figure ). However, treatment of fibroblast cultures with IFN-γ alone or with IFN-γ in combination with IL-1β or TNF-α resulted in a modest but significant increase of membrane-associated DPP IV activity (Figure ). FACS analysis confirmed the slightly increased CD26 expression on IFN-γ-treated and IL-1β-treated fibroblasts (Figure ).
Enhanced levels of CXCR3 ligands in rheumatic disorders
Synovial fluids from patients (n
= 126) with rheumatic diseases including RA, AS, PsA and CA were analysed for their CXCL8 and CXCL10 content by specific ELISAs (Figure ). Compared with CA patients, the median synovial CXCL10 levels were significantly enhanced in patients with RA (P
), in patients with AS (P
) and in patients with PsA (P
). No statistically significant difference in synovial fluid concentrations of CXCL10 was observed between the three types of autoimmune rheumatic disorders. The median CXCL10 concentration for the three types of autoimmune arthritis varied between 10–20 ng/ml, versus <1 ng/ml for CA. The mean level of synovial CXCL10 in the autoimmune arthritis patients was comparable with that measured in septic arthritis [11
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 (more ...)
In contrast to CXCL10, synovial CXCL8 concentrations were only significantly (P < 0.05) enhanced in RA patients, and not in PsA or AS patients, in comparison with CA patients (Figure ). This indicates that not the neutrophil chemoattractant CXCL8, but rather the Th1 lymphocyte chemoattractant CXCL10 is implicated in PsA and in AS, whereas none of the chemokines are associated with CA. No correlation was detected between CXCL8 and CXCL10 levels nor between CXCL8 or CXCL10 and serum C-reactive protein levels (data not shown).