Differential survival of leukocyte subsets mediated by synovial, bone marrow, and skin fibroblasts: site-specific versus activation-dependent survival of T cells and neutrophils

Abstract

Objective: Synovial fibroblasts share a number of phenotype markers with fibroblasts derived from bone marrow. In this study we investigated the role of matched fibroblasts obtained from 3 different sources (bone marrow, synovium, and skin) to test the hypothesis that synovial fibroblasts share similarities with bone marrow-derived fibroblasts in terms of their ability to support survival of T cells and neutrophils.

Methods: Matched synovial, bone marrow, and skin fibroblasts were established from 8 different patients with rheumatoid arthritis who were undergoing knee or hip surgery. Resting or activated fibroblasts were cocultured with either CD4 T cells or neutrophils, and the degree of leukocyte survival, apoptosis, and proliferation were measured.

Results: Fibroblasts derived from all 3 sites supported increased survival of CD4 T cells, mediated principally by interferon-beta. However, synovial and bone marrow fibroblasts shared an enhanced site-specific ability to maintain CD4 T cell survival in the absence of proliferation, an effect that was independent of fibroblast activation or proliferation but required direct T cell-fibroblast cell contact. In contrast, fibroblast-mediated neutrophil survival was less efficient, being independent of the site of origin of the fibroblast but dependent on prior fibroblast activation, and mediated solely by soluble factors, principally granulocyte-macrophage colony-stimulating factor.

Conclusion: These results suggest an important functional role for fibroblasts in the differential accumulation of leukocyte subsets in a variety of tissue microenvironments. The findings also provide a potential explanation for site-specific differences in the pattern of T cell and neutrophil accumulation observed in chronic inflammatory diseases.

Figure 1

Structural and cytokine expression profiles of synovial- and bone marrow–derived fibroblasts as compared with dermal fibroblasts. A, Morphologic findings (at confluence) in a matched set of resting synovial, bone marrow, and skin fibroblasts. Cells were grown at passage 5 in glass-bottom chamber slides and then visualized by differential interference contrast (DIC). The DIC images of cells overlaid with fluorescence images (right panels) show staining for fibronectin (red) and nuclear staining with 4′,6-diamidino-2-phenylindole (blue) (original magnification × 20 in left panels; × 32 in right panels). B, Surface vascular cell adhesion molecule 1 (VCAM-1; CD106) expression of resting fibroblasts was quantified by immunofluorescence microscopy. Results are the combined mean and SD number of positively staining cells per field for all 8 matched samples. C and D, Fibroblasts (from 3 patients) were cultured in the absence or presence of tumor necrosis factor α (TNFα) for 24 hours. Supernatants were collected after a further 24 hours and production of interleukin-6 (IL-6) (C) and CCL2 (D) was analyzed quantitatively by enzyme-linked immunosorbent assay. Results are the mean and SD of duplicate assays of combined fibroblasts from 3 patients. * = P < 0.05; ** = P < 0.01. ns = not significant.

Figure 2

Support of site-specific survival of CD4 T cells by resting fibroblasts in the absence of T cell proliferation. A, Increased survival of CD4 T cell lines cocultured for 3 days with synovial, bone marrow, and dermal fibroblasts as compared with that in cultures without fibroblasts (control) or with interleukin-2 (IL-2) (as a positive control). B, Association of T cell survival with the decreased rates of apoptosis, as measured by caspase 3 activation. Results in A and B are the mean and SEM from 8 individuals. At least 2 separate experiments with triplicate assays were performed using matched synovial, bone marrow, and skin fibroblasts at identical passage. * = P < 0.05; ** = P < 0.01; *** = P < 0.001. ns = not significant. C, Labeling of T cells with 5-(and 6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE) demonstrating the absence of proliferation on cocultures with fibroblasts as compared with positive control T cells treated with IL-2 plus phytohemagglutinin (PHA). D, Lack of association between T cell survival and fibroblast proliferation. Results are depicted as a scatter plot of T cell survival against the proliferation index (ratio of mean triplicate fibroblast cell counts at day 6 compared with day 0). Each point represents an individual cell strain (squares = bone marrow; triangles = synovium; circles = skin). The r value is the Spearman’s correlation coefficient.

Figure 3

Support of neutrophil survival by resting fibroblasts, with no site specificity. A, Neutrophil survival is modestly increased (as compared with control cultures) on cocultures with all fibroblasts (P < 0.01), but no difference between sites of origin of the fibroblasts is evident. Granulocyte–macrophage colony-stimulating factor (GM-CSF) was included as a positive control. B, Increased neutrophil survival is mirrored by decreased apoptosis as measured by mitochondrial 3,3′-dihexyloxacarbocyanine iodide retention (P < 0.05). Results are the mean and SEM from 8 individuals. At least 2 separate experiments with triplicate assays were performed using matched synovial, bone marrow, and skin fibroblasts at identical passage. ns = not significant.

Figure 4

Effect of prior fibroblast activation with proinflammatory cytokines on leukocyte survival. A, Matched synovial, bone marrow, and skin fibroblasts were preactivated with tumor necrosis factor α (TNFα), interleukin-17 (IL-17), interferon-γ (IFNγ) (each at 10 ng/ml), and 1 ng/ml of IL-1β, and then cocultured with CD4 T cells. No significant effect of preactivation on CD4 T cell survival can be seen. IL-2 was included as a positive control. Results are the mean and SEM from 3 independent experiments. B, Following preactivation of matched fibroblasts with TNFα, IL-17, IFNγ, and IL-1β at the same concentrations as in A and coculture with neutrophils, enhanced survival of neutrophils can be seen with all 3 types of fibroblasts. Granulocyte–macrophage colony-stimulating factor (GM-CSF) was included as a positive control. Results are the mean and SEM from 8 individuals in at least 2 separate experiments for each.

Figure 5

Prerequisite of initial cell–cell contact for enhanced T cell survival, and partial mediation by interferon-β (IFNβ). A, CD4 T cells were cocultured with matched fibroblasts from synovium, bone marrow, and skin in the presence or absence of transwells. Interleukin-2 (IL-2) was included as a positive control. B, CD4 T cells were incubated with fibroblasts, fibroblast-conditioned medium (FCM), or medium obtained from whole coculture (full CM). No effect of FCM on T cell survival can be seen, whereas full CM is sufficient to reconstitute site-specific survival. Results are the mean and SEM of 4 independent experiments in A and 3 independent experiments in B. C, CD4 T cells were incubated with full CM in the presence or absence of specific antisera capable of blocking IFNβ activity. IFNβ-specific antisera blocked survival to a similar degree regardless of site of origin. Results are the mean and SEM of 4 independent experiments. * = P < 0.05; ** = P < 0.01.

Figure 6

No requirement for initial cell–cell contact in enhanced neutrophil survival, and partial mediation by granulocyte–macrophage colony-stimulating factor (GM-CSF). A, Peripheral blood neutrophils were incubated with either resting or tumor necrosis factor α (TNFα)–stimulated fibroblasts, or with fibroblast-conditioned medium (FCM) or TNFα-activated FCM prepared from the same fibroblasts. Neutrophil survival induced by TNFα-activated FCM was identical to that induced by direct coculture with resting or stimulated fibroblasts. B, Matched fibroblasts from synovium, bone marrow, and skin were cultured in the absence or presence of TNFα for 24 hours. Supernatants were collected after a further 24 hours and GM-CSF was measured by enzyme-linked immunosorbent assay. * = P < 0.05. C, Peripheral blood neutrophils were incubated with varying amounts of GM-CSF, and survival of neutrophils was determined. D, Peripheral blood neutrophils were incubated with medium alone, GM-CSF, or TNFα-activated FCM, some of which had been depleted using antibodies against GM-CSF or isotype-matched irrelevant antibodies. Depletion using antibodies to GM-CSF resulted in a mean inhibition of enhanced neutrophil survival of ~54% (P = 0.029). As a positive control, GM-CSF was added to neutrophils either before or after depletion with anti–GM-CSF antibodies. * = P < 0.05; ** = P < 0.01. Results are the mean and SEM of 3 independent experiments.