Constitutive expression of IL-22 in the human intervertebral disc and its reduction by exposure to pro-inflammatory cytokines in vitro.

The importance of cytokines in disc degeneration is well recognized. Little is known about IL-22 expression in the human intervertebral disc. We investigated IL-22 immuno-localization in disc tissue, and molecular expression and production of IL-22 by annulus cells cultured in three-dimensional (3D) culture. We examined human disc tissue using immunohistochemistry and we cultured isolated annulus cells in 3D to analyze IL-22 expression and production, and its receptor, IL-22R, in conditioned media. Ingenuity pathway analysis (IPA) also was used to identify significant gene expression networks within the molecular data. IL-22 and IL-22R were immunolocalized in many cells in the human outer and inner annulus; fewer cells exhibited localization in the nucleus. Three-dimensional culture of annulus cells demonstrated production of IL-22 in conditioned media; exposure to IL-1ß or TNF-α significantly reduced IL-22 levels. Significant decreases also were identified in conditioned media assayed for IL-22R in TNF-α treated cells. IPA analysis showed that IL-22 ranked among the top canonical pathways. We found constitutive expression and production of IL-22 and IL-22R in the disc, which expands our understanding of the effect of pro-inflammatory cytokines on IL-22 expression and production. Three-dimensional cultured annulus cells exposed to IL-1ß or TNF produced significantly lower levels of IL-22 into their conditioned media compared to levels produced by control cells. Our findings have clinical relevance because of the elevated pro-inflammatory milieu within the degenerating human disc.

The chemokine, CXCL16, and its receptor, CXCR6, are constitutively expressed in human annulus fibrosus and expression of CXCL16 is up-regulated by exposure to IL-1ß in vitro.

Chemokines are an important group of soluble molecules with specialized functions in inflammation. The roles of many specialized chemokines and their receptors remain poorly understood in the human intervertebral disc. We investigated CXCL16 and its receptor, CXCR6, to determine their immunolocalization in disc tissue and their presence following exposure of cultured human annulus fibrosus cells to proinflammatory cytokines. CXCL16 is a marker for inflammation; it also can induce hypoxia-inducible factor 1α (HIF-1α), which is a phenotypic marker of heathy nucleus pulposus tissue. We found CXCL16 and CXCR6 immunostaining in many cells of the annulus portion of the disc. Molecular studies showed that annulus fibrosus cells exposed to IL-1ß, but not TNF-α, exhibited significant up-regulation of CXCL16 expression vs. control cells. There was no significant difference in the percentage of annulus cells that exhibited immunolocalization of CXCL16 in grade I/II, grade III or grade IV/V specimens. The presence of CXCL16 and its receptor, CXCR6, in the annulus in vivo suggests the need for future research concerning the role of this chemokine in proinflammatory functions, HIF-1α expression and disc vascularization.

Increased IL-17 expression in degenerated human discs and increased production in cultured annulus cells exposed to IL-1ß and TNF-α.

IL-17 is expressed in a number of tissues including the intervertebral disc, where it exerts strong inflammatory properties. We evaluated IL-17 using immunolocalization in herniated and non-herniated human discs, IL-17 gene expression, and the production of IL-17 by annulus cells cultured in three dimensions in the presence of IL-1ß or TNF-α. There was no difference in the percentage of IL-17 positive cells in annulus or nucleus in herniated vs. non-herniated disc specimens. Molecular studies confirmed expression of IL-17 in disc tissue, with significantly increased expression in more degenerated discs; there was no difference in expression between herniated vs. non-herniated discs. Exposure to IL-1ß or TNF-α resulted in significantly greater production of IL-17. Our findings expand understanding of IL-17 production by disc cells and reveal the importance of non-canonical IL-17 production in the disc. Significantly greater expression of IL-17 in more degenerated discs adds to our understanding of the changes in disc cell function with advancing stages of disc degeneration.

Interleukin 1-beta upregulates brain-derived neurotrophic factor, neurotrophin 3 and neuropilin 2 gene expression and NGF production in annulus cells.

The relationship between disc cells, nerves and pain production in the intervertebral disc is poorly understood. Neurotrophins, signaling molecules involved in the survival, differentiation and migration of neurons, and neurite outgrowth, are expressed in non-neuronal tissues including the disc. We hypothesized that three-dimensional exposure of human disc cells to the proinflammatory cytokine IL-1ß in vitro would elevate neurotrophin gene expression levels and production of nerve growth factor (NGF). Cells isolated from Thompson grade III and IV discs were cultured for 14 days under control conditions or with addition of 10(2) pM IL-1ß; mRNA was isolated and conditioned media assayed for NGF content. IL-1ß exposure in three-dimensional culture significantly increased expression of neurotrophin 3, brain-derived neurotrophic factor, and neuropilin 2 compared to controls. IL-1ß-exposed cells showed significantly increased NGF production compared to controls. Findings support our hypothesis, expand previous data concerning expression of neurotrophins, and provide the first documented expression of neurotrophin 3 and neuropilin 2. Our results have direct translational relevance, because they address the primary clinical issue of low back pain and open the possibility of novel analgesic therapies using specific small-molecular antagonists to neurotrophins.

Human adipose-derived mesenchymal stem cells: serial passaging, doubling time and cell senescence.

Adult adipose-derived mesenchymal stem cells (AD-MSC) are very interesting to our research group because they are easy to harvest, they are abundant in humans, and they have potential clinical applications in autologous cell therapy for disc degeneration. We examined these cells through sequential serial passages to assess osteogenic and chondrogenic capabilities, mean doubling time and cell senescence. Osteogenic and chondrogenic potencies were maintained through 13 passages. Mean passage doubling time increased significantly with increasing passage number. When donor age was evaluated, passages 1-4 from older donors had significantly longer doubling times compared to cells from younger donors. Passages 5-11 showed similar findings when analyzed by donor age. The mean percent senescence increased significantly with cell passaging, rising from 0% at passage 1 to 3.4% at passage 13. These novel data suggest that caution should be exercised when using AD-MSC with long passage times.

Periostin is expressed by cells of the human and sand rat intervertebral discs.

Periostin, a matricellular protein in the fasciclin family, is expressed in tissues subjected to constant mechanical stress. Periostin modulates cell-to-extracellular matrix interactions and can bind to collagen, fibronectin, tenascin-C and several integrins. Our objective was to evaluate whether periostin is expressed in the human intervertebral disc. Immunohistochemical localization of periostin was carried out in tissue of human lumbar discs and lumbar discs of the sand rat (Psammomys obesus). Human discs also were examined for periostin gene expression. Immunohistochemical localization demonstrated periostin in the cytoplasm of annulus and nucleus cells, and occasionally in the surrounding pericellular and interterritorial extracellular matrix. Periostin distribution in the human disc was distinctive. Outer annulus contained the highest proportion of periostin-positive cells (88.8%), whereas inner annulus contained only 61.4%. The nucleus pulposus contained the fewest periostin-positive cells (18.5%). There was a significant negative correlation between the percentage of cells positive for periostin in the inner annulus and subject age. Periostin gene expression in the human disc also was confirmed using molecular microarray analysis. Because work by others has shown that periostin plays an important role in the biomechanical properties of other connective tissues (skin, tendon, heart valves), future research is needed to elucidate the role of periostin in disc, loading, aging and degeneration.

Effects of very high antibiotic concentrations on human intervertebral disc cell proliferation, viability, and metabolism in vitro.

STUDY DESIGN: Four antibiotics commonly used during spinal surgery (cefazolin, gentamycin, cefamandole, and vancomycin) were tested for their effects on cultured human disc cells from the anulus. OBJECTIVE: To determine the viability, proliferation, and metabolism of cells cultured from the human anulus after they were exposed to four antibiotics. SUMMARY OF BACKGROUND DATA: Previous studies concerning the effect of antibiotics on the disc have used animal models or explanted discs, but little is understood about the effect of antibiotics on the proliferation, viability, and metabolism of cells from the anulus. METHODS: In this study, 3H-thymidine incorporation, trypan blue exclusion, and cell metabolism were determined using cells from the human anulus grown in monolayer culture. The latter measurement used a cytosensor microphysiometer to monitor the rate at which cells acidified their microenvironment, an event that is proportional to cellular metabolism because it reflects the excretion over time of acidic products such as lactic acid from glycolysis and CO2 from cellular respiration. RESULTS: After 48 hours of antibiotic exposure, cell viability was significantly lower as a result of all four antibiotics at the highest concentration tested. Cell proliferation was lower after exposure to cefazolin and cefamandole. During a 6-hour antibiotic exposure, anulus cells in the highest concentration of cefamandole or vancomycin displayed a significantly decreased rate of cell metabolism. CONCLUSIONS: These findings show that high doses of antibiotics can have direct, deleterious effects on cultured disc cell survival, cell proliferation, and metabolic rates. Discitis is a serious primary or postoperative complication that often requires prolonged antibiotic treatment. Studies such as the current investigation with cultured cells from the anulus show the importance of a greater understanding concerning antibiotic effects on disc cell proliferation and metabolism.

Detection of apoptotic cells in vitro: a practical standardized experimental protocol using human fibroblasts for initial laboratory studies.

This technical report presents a practical experimental protocol using human fibroblasts that is useful for initiating in situ methods for detection of apoptotic cells in vitro. The protocol details the growth of cells in multichamber wells, and includes a negative control, two positive controls, and untreated fibroblasts to be evaluated for apoptosis localization. Technical tips on handling of solutions, cell culture designs, and separation of slides by treatment are valuable steps for the laboratory beginning such studies. The methods proposed are both time and cost effective.

Characterization of an unusual fluoride-resistant Streptococcus mutans isolate.

A fluoride-resistant Streptococcus mutans isolate NCH105 was characterized and compared with wild-type strain UA130. The growth and lactic acid production of strain NCH105 were found to be unaffected by the presence of fluoride at the initial medium pH values of 6.5 and 6. In addition, NCH105 was found to be capable of lowering the pH of the medium to approximately 5.5, which is the critical level where enamel demineralization begins in vivo. Lactic acid production, glucose uptake at pH 6.5 and 6, as well as ATPase activity at pH 5 were found to be unaffected by fluoride. Finally, strain NCH105 is capable of binding as well as the wild-type bacterium to artificial tooth pellicles. These results are unusual when compared with previously isolated fluoride-resistant mutants and suggest that NCH105 may have the ability to colonize the tooth surface and initiate dental caries.