Hilary P. Benton

Cytokines and their receptors

Cytokines act via receptor-mediated pathways to influence the regulation of both immune and non-immune cells. This review will discuss some of the most important developments over the past year which have contributed to the elucidation of the mechanisms of cell activation by these molecules.

ATP and UTP activate calcium-mobilizing P2U-like receptors and act synergistically with interleukin-1 to stimulate prostaglandin E2 release from human rheumatoid synovial cells

OBJECTIVE To pharmacologically and functionally characterize calcium-mobilizing purine receptors on adherent human rheumatoid synovial cells. METHODS Fura-2-loaded synovial cells were screened for changes in cytosolic calcium concentration after the addition of purine receptor agonists. Release of interleukin-1 (IL-1) and prostaglandin E2 (PGE2) was assessed by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay, respectively. The effect of IL-1 prestimulation on purine-mediated PGE2 release was determined. RESULTS ATP (1-100 microM) and UTP (1-100 microM), but not 2-methylthio-ATP or adenosine, stimulated mobilization of calcium from intracellular stores in synovial cells. ATP and UTP stimulated a small, but significant, increase in PG release from resting synoviocytes and a dramatic increase in PG release from synoviocytes prestimulated with recombinant human IL-1alpha. Neither ATP nor UTP stimulated synoviocyte release of IL-1 as measured by specific ELISA. The effects of ATP and UTP on PG secretion were mimicked by phorbol 12-myristate 13-acetate and thapsigargin, and blocked by BAPTA buffering of cytosolic calcium. CONCLUSION Adherent human rheumatoid synovial cells mobilize intracellular calcium via a P2U-like purine receptor. P2U receptor agonists stimulate PGE2 release from synoviocytes, an effect that is greatly enhanced by IL-1alpha prestimulation and blocked by intracellular calcium buffering.

Two-photon excitation laser scanning microscopy of human, porcine, and rabbit nasal septal cartilage

Two-photon excitation laser scanning microscopy (TPM) was used to image human, porcine, and rabbit nasal septal cartilage. TPM provides optical sections of thick tissue specimens in situ without the use of exogenous dyes or need for tissue fixation. The cartilage tissue was imaged using near-infrared light generated by a mode-locked titanium/sapphire laser that was raster-scanned and coupled to an inverted microscope. Absorption of two photons by endogenous molecules and subsequent fluorescence was filtered to specific spectral bandwidths and detected with photomultiplier tubes. Two-photon stimulated fluorescence was detected with photomultiplier tubes optimized to specific spectral bandwidths. Signal intensity corresponds to the concentration of fluorophores, principally NADH, NADPH, and flavoproteins hence providing a means of redox imaging the cellular metabolic state. Specimens were scanned from the surface to a depth of about 150 microm. Image size was 50 x 50 microm with a diffraction limited pixel size of 0.4 microm. Cell membranes, nuclei, and matrix structures were identified in human, pig, and rabbit tissues. TPM provides a means to study three dimensional chondrocyte structure and matrix organization in situ at substantial depths, and permits longitudinal examination of cultured tissue explants without the need for exogenous dyes, tissue preparation, or fixation.

Influence of Alginate Polysaccharide Composition and Culture Conditions on Chondrocytes in Three-Dimensional Culture

Alginate-embedded chondrocytes have been used for experimental analysis of cartilage matrix metabolism and as a potential bioartificial system for repairing cartilage defects. Alginates are linear copolymers composed of 1,4-linked beta-n-mannuronic acid and 1,4 linked alpha-L-guluronic acid, which occur as regions made up exclusively of one unit or the other, or as regions in which the monomers approximate an alternating sequence. Data presented here demonstrate that the mannuronic to guluronic acid (M/G) ratio and molecular weight of the alginate utilized effects the tissue-culture handling properties of the resultant gel and the activity of embedded chondrocytes. In experiments comparing chondrocyte survival and matrix synthesis, optimal conditions were obtained with an alginate of both high mannuronic acid content and high molecular weight. Chondrocytes survived and proliferated when maintained in unsupplemented media, in media supplemented with fetal calf serum, and in media supplemented with the defined serum replacement ITS+ (6.25 microg/ml insulin, 6.25 microg/ml transferrin, 6.25 ng/ml selenous acid, 1.25 mg/ml bovine serum albumin, 5.35 microg/ml linoleic acid). High cell survival rate and increase in cell number was obtained in the absence of serum. In contrast, long-term matrix synthesis and deposition required media supplementation as indicated by uptake of [(35)S]sulfate into glycosaminoglycans and by immunofluorescence using antibodies specific for cartilage matrix molecules.

Characterization of age- and location-associated variations in the composition of articular cartilage from the equine metacarpophalangeal joint

Abstract Objective: To characterize the impact of age, gender, location and individual animal variation on the composition of articular cartilage from the metacarpophalangeal joint of horses. Design: Cartilage specimens were obtained from the metacarpophalangeal joints of 28 male, female and castrated male horses ranging in age from one day to 27 years of age. Cartilage samples from the distal metacarpus, proximal first phalanx and proximal sesamoids were analyzed separately. Chondrocyte number, DNA content, proteoglycan concentration and total collagen content were determined for each animal and joint location. Results: Age and joint location had a significant effect on chondrocyte number and DNA content with higher cell counts and DNA content detected in cartilage from the youngest age groups and in cartilage from the metacarpus and proximal sesamoids. The influence of age on chondrocyte numbers was not significant in horses over two years of age. Both age and joint location also influenced total proteoglycan and collagen content. Lower proteoglycan and collagen concentrations were detected in younger horses, and cartilage from the metacarpus had lower proteoglycan and collagen concentrations than that from other joint locations. Gender did not appear to influence chondrocyte number or matrix content of equine articular cartilage. However, there was significant residual variation in cellularity, proteoglycan levels and collagen content between individual animals that could not be explained by the signalment factors considered in this study. Conclusions: Future studies examining equine articular cartilage should avoid direct morphologic comparisons between animals of different ages, and any comparisons made between individuals should be interpreted cautiously. In addition, in vitro tissue culture models should avoid the use of cartilage pooled from different animals or from different locations within the same joint.

Histology of porcine nasal cartilage grafts following Nd:YAG (1320 nm) laser mediated reshaping : Effects of sequential irradiation

Mechanically deformed morphologic cartilage grafts undergo temperature dependent stress relaxation during sustained laser irradiation resulting in stable shape changes. In this study, the porcine nasal septal cartilage specimens were evaluated histologically following laser mediated reshaping using H&E. Cartilage specimens were irradiated with light emitted from a Nd:YAG laser (25 W/cm2, 1 = 1.32 mm) while recording simultaneously radiometric surface temperature, internal stress, and backscattered light intensity from a probe laser. Each specimen received one, two, or three sequential laser exposures. The duration of each exposure was determined from real-time measurements of characteristic changes in backscattered light intensity that correlate with accelerated stress relaxation. A five minute time interval between each laser exposures allowed the cartilage specimen to return to thermal equilibrium. Specimens were then fixed in formalin, serially dehydrated in ethanol, embedded in paraffin, and sectioned with a microtome for histologic examination using light microscopy. Large variation in native tissue histology was observed among individual tissue samples, and vascular were identified in several specimens. Large lacunae with shrunken chondrocytes were identified along with cells with pyknotic nuclei, although these histologic observations did not correlate with the degree of laser exposure. These observations are discussed.

Nonlinear Optical Microscopy and Spectroscopy of Articular Cartilage

Nonlinear optical microscopy is used to image living articular cartilage in situ without exogenous stains or dyes. Endogenous nonlinear optical signals may be used for image segmentation and to evaluate articular cartilage matrix health.