Interactive cytokine regulation of synoviocyte lubricant secretion

Abstract

Cytokine regulation of synovial fluid (SF) lubricants, hyaluronan (HA), and proteoglycan 4 (PRG4) is important in health, injury, and disease of synovial joints, and may also provide powerful regulation of lubricant secretion in bioreactors for articulating tissues. This study assessed lubricant secretion rates by human synoviocytes and the molecular weight (MW) of secreted lubricants in response to interleukin (IL)-1beta, IL-17, IL-32, transforming growth factor-beta 1 (TGF-beta1), and tumor necrosis factor-alpha (TNF-alpha), applied individually and in all combinations. Lubricant secretion rates were assessed using ELISA and binding assays, and lubricant MW was assessed using gel electrophoresis and Western blotting. HA secretion rates were increased approximately 40-fold by IL-1beta, and increased synergistically to approximately 80-fold by the combination of IL-1beta + TGF-beta1 or TNF-alpha + IL-17. PRG4 secretion rates were increased approximately 80-fold by TGF-beta1, and this effect was counterbalanced by IL-1beta and TNF-alpha. HA MW was predominantly <1 MDa for controls and individual cytokine stimulation, but was concentrated at >3 MDa after stimulation by IL-1beta + TGF-beta1 + TNF-alpha to resemble the distribution in human SF. PRG4 MW was unaffected by cytokines and similar to that in human SF. These results contribute to an understanding of the relationship between SF cytokine and lubricant content in health, injury, and disease, and provide approaches for using cytokines to modulate lubricant secretion rates and MW to help achieve desired lubricant composition of fluid in bioreactors.

FIG. 1.

Synovial joints may be modeled theoretically with a compartmental approach, and experimentally in a bioreactor with key components that include lubricant-secreting cell types, a SF compartment, and a lubricant-retaining semipermeable membrane. Chemical and mechanical stimuli are key parameters that may regulate lubricant secretion and MW, and affect lubricant composition in native and bioengineered SF. MW, molecular weight; SF, synovial fluid. Color images available online at www.liebertonline.com/ten.

FIG. 2.

Regulatory effects of the individual cytokines IL-1β, IL-17, IL-32, TGF-β1, and TNF-α at a range of concentrations on (A) proliferation, (B) HA secretion, and (C) PRG4 secretion by human synoviocytes. Mean ± SEM, n = 5–6; *p < 0.05. HA, hyaluronan; PRG4, proteoglycan 4; IL, interleukin; TGF-β1, transforming growth factor-beta 1; TNF-α, tumor necrosis factor-alpha; SEM, standard error of the mean.

FIG. 3.

Regulatory effects of different combinations of the cytokines IL-1β, IL-17, IL-32, TGF-β1, and TNF-α (applied at maximum individual stimulation doses) on (A) proliferation, (B) HA secretion, and (C) PRG4 secretion by human synoviocytes. Mean ± SEM, n = 6; *p < 0.05 for individual effect; **p < 0.05 for interactive effect. (+) signs indicate cytokines present in the culture media.

FIG. 4.

(A) Observation of HA in conditioned medium samples from selected regulatory cytokine treatments in the MW range of 0.1–7.0 MDa, using agarose gel electrophoresis technique. Plus (+) signs indicate the negative control samples that were subjected to HA digestion with Streptomyces hyaluronidase. (B) Percentage of total HA in the MW ranges of >3, 1–3, and <1 MDa, averaged from relative intensities of samples in (A), mean ± SEM, n = 2–4; *p < 0.05 versus both control and TGF-β1; ^#p < 0.05 versus IL-1β.

FIG. 5.

(A) Relative HA intensity profiles in the MW range of 0.1–7.0 MDa for all conditioned medium samples (pooled from n = 6) and normal human SF. (B) Percentage of total HA in the MW ranges of >3, 1–3, and <1 MDa, calculated from relative intensities of samples in (A). (+) signs indicate cytokines present in the culture media. Color images available online at www.liebertonline.com/ten.

FIG. 6.

Observation of PRG4 from conditioned medium with selected individual and combination cytokine treatments and also human SF, using techniques of gel electrophoresis, Western blotting, and chemiluminescent detection. (+) signs indicate cytokines present in the culture media.