Rabbit articular cartilage defects treated with autologous cultured chondrocytes

Adult New Zealand rabbits were used to transplant autologously harvested and in vitro cultured chondrocytes into patellar chondral lesions that had been made previously and were 3 mm in diameter, extending down to the calcified zone. Healing of the defects was assessed by gross examination, light microscope, and histological-histochemical scoring at 8, 12, and 52 weeks. Chondrocyte transplantation significantly increased the amount of newly formed repair tissue compared to the found in control knees in which the lesion was solely covered by a periosteal flap. In another experiment, carbon fiber pads seeded with chondrocytes were used as scaffolds, and repair significantly increased at both 12 and 52 weeks compared to knees in which scaffolds without chondrocytes were implanted. The histologic quality scores of the repair tissue were significantly better in all knees in which defects were treated with chondrocytes compared to knees treated with periosteum alone and better at 52 weeks compared to knees in which defects were treated with carbon scaffolds seeded with chondrocytes. The repair tissue, however, tended to incomplete the bonding to adjacent cartilage. This study shows that isolated autologous articular chondrocytes that have been expanded for 2 weeks in vitro can stimulate the healing phase of chondral lesions. A gradual maturation of the hyalinelike repair with a more pronounced columnarization was noted as late as 1 year after surgery.     

Repair of large full-thickness articular cartilage defects with allograft articular chondrocytes embedded in a collagen gel

Full-thickness articular cartilage defects are a major clinical problem; however, presently there is no treatment available to regeneratively repair these lesions. The current therapeutic approach is to drill the base of the defect to expose the subchondral bone with its cells and growth factors. This usually results in a repair tissue of fibrocartilage that functions poorly in the loaded joint environment. The use of phenotypically appropriate chondrocytes embedded in a collagen gel delivery vehicle may provide a method that could be used to repair full-thickness articular cartilage defects with functionally satisfactory hyaline cartilage. Allograft articular chondrocytes embedded in a type I collagen gel were transplanted into large (6 x 3 x 3 mm), full-thickness articular cartilage defects in condylar and patellar weight-bearing surfaces to develop clinically applicable methods to repair articular cartilage defects. Chondrocytes were isolated from the articular cartilage of 4-week-old New Zealand rabbits and embedded in type I collagen gels. This composite was transplanted into a full-thickness defect on the medial femoral condyle and patellar groove of adolescent host rabbits. The repair cartilage was assessed histologically by a semiquantitative scoring system and biomechanically with a microindentation technique of specimens 4-48 weeks after chondrocyte transplantation. Defects in both locations were repaired with histologically apparent hyaline cartilage observed from as early as 4 weeks until 48 weeks after transplantation. The repair cartilage in the medial femoral condyle was more irregular than in the patellar groove, but in all other respects was similar. The grafted tissue did not remodel and differentiate into the morphological zones seen in normal articular cartilage. No tidemark or subchondral bony plate formed even 48 weeks after transplantation. Biomechanically, the repaired cartilage demonstrated indentation values similar to normal articular cartilage 12 weeks after transplantation and remained the same 48 weeks after transplantation. By contrast, the control (i.e., empty) defects healed with tissue that exhibited very poor metachromatic staining and exhibited very high indentation values. Incomplete bonding of the repair tissue to the normal cartilage was seen, and the surface was significantly irregular with major discontinuities. These observations provide the basis for considering the use of allograft articular chondrocytes to repair articular cartilage defects in the weight-bearing regions of the knee.     

Metallothionein induction in calcifying growth plate cartilage chondrocytes

Metallothionein (MT) induction was studied in mineralizing cultures of chicken growth plate chondrocytes and quantitated using a Cd-saturation assay. In serum free media, MT induction was observed for Cd concentrations of 0.1 microM and greater and at Zn concentrations of 100 microM and greater. Supplementation of culture media with cysteine and/or methionine resulted in higher levels of MT induction and reduced toxicity during Cd exposure. Maximum MT induction appeared to coincide with the earliest culture stages during which important enzymes and matrix components are being synthesized. Of non-metal MT inducers tested, sodium butyrate caused a low level induction of MT while interleukin-1 had no effect on basal MT levels. 1,25-dihydroxyvitamin D increased MT induction. The steroid hormone dexamethasone caused a reduction in basal and induced MT levels. These findings suggest that MT regulation in growth plate chondrocytes differs significantly from what is known in other cell types and that this difference may be related to the mineralization of this tissue.     

Differential expression of aggrecanase and matrix metalloproteinase activity in chondrocytes isolated from bovine and porcine articular cartilage

The release of aggrecan catabolites from cartilage is an early event in the pathogenesis of degenerative joint diseases. The enzymes involved in this process are unknown, controversial, and the subject of intense investigation. In this paper we have utilized a recombinant substrate containing the interglobular domain (IGD) of aggrecan to study specifically aggrecanase versus matrix metalloproteinase (MMP) catabolism in this domain of aggrecan. Our studies have shown that (i) there are species differences in the expression of latent versus active MMP activity on the aggrecan IGD; (ii) interleukin-1alpha exposure induces both aggrecanase and MMP activities, whereas retinoic acid induces only aggrecanase activity and inhibits the MMP activity on the aggrecan IGD; (iii) activators of latent MMP activity (p-aminophenylmercuric acetate and trypsin) significantly reduce aggrecanase activity; (iv) the time course of the appearance of aggrecanase versus the MMP catabolism of aggrecan IGD differs; (v) aggrecanase is a protease with metalloprotease characteristics; however (vi) the physiological (tissue) inhibitors of MMPs show weak inhibition (TIMP-1) or no inhibition (TIMP-2) of aggrecanase activity. Collectively, these studies show that aggrecanase and MMP catabolism of the aggrecan IGD are independent and uncoupled.     

Delta-1 negatively regulates the transition from prehypertrophic to hypertrophic chondrocytes during cartilage formation

Endochondral bone development begins with the formation of a cartilage template. Chondrocytes within this template undergo a progressive program of maturation from proliferative to prehypertrophic chondrocytes to hypertrophic chondrocytes. The progression of cells through these steps of differentiation must be carefully controlled to ensure coordinated growth. Because the Delta/Notch signaling system is known to regulate cell fate choices, we sought to determine if these molecules might be involved in the progressive cell fate decisions that chondocytes undergo. Here we demonstrate in the chick that Delta/Notch signaling negatively regulates progression from the prehypertrophic to hypertrophic state of differentiation. Delta-1 is expressed specifically in the hypertrophic chondrocytes while Notch-2 is expressed in chondrocytes at all stages. Misexpression of Delta-1 using a replication-competent retrovirus blocks chondrocyte maturation. Prehypertrophic cells form normally but do not undergo differentiation to hypertrophic cells, resulting in shortened skeletal elements that lack ossification. We conclude that Delta-1 acts during chondrogenesis to inhibit the transition from prehypertrophic chondrocytes to hypertrophic chondrocytes, thus defining a novel mechanism for the regulation of the chondrocyte maturation program. In addition, these results reveal a new role for Delta/Notch signaling in regulating the progression to a terminally differentiated state.     

Regulation of cartilage oligomeric matrix protein synthesis in human synovial cells and articular chondrocytes

OBJECT: The relationship between glial fibrillary acidic protein (GFAP) expression and glial tumor cell behavior has not been well defined. The goal of this study was to examine this relationship further. METHODS: To investigate the relationship between GFAP expression and glial tumor cell behavior, the authors isolated clones from the human glioblastoma cell line, U-373MG, according to their level of GFAP expression. Immunochemical analysis demonstrated that one clone had consistently low GFAP expression (approximately 93% of cells were GFAP negative), whereas a second clone had consistently high GFAP expression (approximately 80% of the cells were GFAP positive). The structure, population doubling time, saturation density, anchorage-independent growth, migratory rate, and invasive potential of these two clones were determined in relation to their level of GFAP expression. Morphologically, both clones were composed of ameboid as well as stellate components. Although the population doubling times of the two clones were equally rapid, the clone with low GFAP expression demonstrated a slightly higher saturation density compared with the clone with high GFAP expression. In an anchorage-independent environment (soft agar), a greater difference in growth characteristics was noted between the two clones: the high-expression clone formed more colonies and these colonies were compact, well defined, and spherical, whereas the low-expression clone formed predominantly smaller, two-dimensional colonies with vague boundaries and isolated cells or groups of cells at the periphery. In contrast to these minor differences between the clones, the low-expression clone showed a markedly increased migratory rate and invasive potential compared with the high-expression clone. Therefore, the clone with reduced GFAP expression appeared more aggressive, demonstrating decreased contact inhibition, increased migratory rate, and increased invasive potential. CONCLUSIONS: These results suggest a direct correlation between GFAP expression and some measures of aggressive tumor growth and transformation properties.     

Effect of static compression on proteoglycan biosynthesis by chondrocytes transplanted to articular cartilage in vitro

Mutant mice that lack either protein zero (P0) or connexin 32 (Cx32) were generated previously to investigate the function of these myelin proteins in peripheral nerves and to assess the value of these mice as animal models for hereditary human peripheral neuropathies. Mice that are completely devoid of P0 expression (P0(+/0)) show a complex phenotype that is characterized by hypomyelination, compromised myelin compaction, and degeneration of myelin and axons early in life. In contrast, young mouse mutants that have retained one wild-type allele of the P0 gene (P0(+/0)) reveal morphologically normal myelin but start to develop signs of demyelination and remyelination at 4 months of age. A similar late-onset myelin deficiency was observed in Cx32-deficient mice (Cx32(0/0)). We have now generated mice deficient for Cx32 and P0. In animals that lack both proteins (Cx32(0/0)/P0(0/0), the phenotype is morphologically identical to mice that solely lack P0. Animals that lack Cx32 and carry one functional P0 allele (Cx32(0/0/P0(+/0)) revealed demyelination and remyelination as evidenced by thin myelin and Schwann cell onion bulb formation already at the age of 4 weeks, a time point when no pathology was observed in the single mutants. These morphological deficits were also more prominent in 4-month-old Cx32(0/0)/P0(+/0)animals compared to the single mutants. Our data support the view that Cx32 and P0 are crucial molecules for the maintenance of myelin. Furthermore, the function of Cx32 in the peripheral nervous system appears to be largely dispensable when myelin compaction is impaired.     

Delayed formation of proteoglycan aggregate structures in human articular cartilage disease states

Proteoglycans are major components of many extracellular matrices. In cartilage, they provide reversible resistance to compression and exist as molecules with molecular weights (MWs) of 1-3 x 10(6). There is a central protein core of MWs approximately 2 x 10(5) (refs 1, 2) with specialized subregions, one containing mainly the chondroitin sulphate chains, another most of the keratan sulphate chains, and a third is a largely globular structure interacting specifically with both hyaluronic acid and a link protein to form stable aggregate structures such as those identified in human articular cartilage. In embryonic and tissue culture systems, proteoglycans are isolated as aggregate structures in as little as 5--10 min after synthesis (sulphation) with no nonaggregating precursor detected. However, Heineg?rd and Hascall have characterized the small proportion of nonaggregating proteoglycan present in bovine nasal septum cartilage and found that it contained more peptide than the aggregating proteoglycan. Work by Upholt et al. has suggested that the MW of unprocessed protein core, synthesized by a wheat-germ translating system from chick sternal cartilage mRNA, is approximately 340,000, leaving open the possibility of intermediates. I report here the presence, in some human cartilages, of a proteoglycan population that initially will not aggregate with the hyaluronic acid but subsequently can be chased into aggregate.     

Magnetic resonance imaging of articular cartilage: ex vivo study on normal cartilage correlated with magnetic resonance microscopy

PURPOSE: The purpose of this study was to determine the effects of a school-based, low-volume strength training program on energy expenditure, strength, and physical fitness in obese prepubertal girls. METHODS: A longitudinal, 5-month strength training exercise program was undertaken by healthy, obese (> 95th percentile weight-for-height, N = 11) girls age 7-10 yr. The following were measured: strength by the one-repetition maximum test; fitness (VO2peak) by a treadmill exercise test; resting metabolic rate (RMR), 24-h sedentary energy expenditure (SEE), and sleeping metabolic rate (SMR) by room respiration calorimetry; and total energy expenditure (TEE) by the doubly labeled water method. Physical activity energy expenditure (AEE) was calculated as TEE-(RMR + 0.1.TEE) and physical activity level (PAL) as TEE/RMR. An age-matched, nonoverweight control group was measured for (VO2peak) and RMR over the same time period. RESULTS: Strength increased by 19.6 and 20.0% in the upper and lower body (P < 0.01), respectively. (VO2peak) (mL.min-1) increased in both groups over time (P < 0.05), but not when covaried for fat-free mass (FFM) or weight. After adjusting for FFM or weight, RMR did not change, but SMR and 24-h SEE decreased significantly in the exercise group. There were no changes in nonprotein respiratory quotient or substrate oxidation. No changes in TEE, AEE, and PAL occurred, either unadjusted or adjusted for FFM or weight. CONCLUSION: This long-term, school-based, low-volume strength training program favorably increases strength in obese prepubertal girls but does not increase their daily energy expenditure.     

Osteoarthritic synovial fluid and synovium supernatants up-regulate tumor necrosis factor receptors on human articular chondrocytes

Microglial reactivity associated with induction of MHC class II (HLA-DR) antigen is a sensitive indicator for pathological events in the CNS. To assess the response of glial cells after lesions of supraspinal descending tracts, HLA-DR, CD68 and GFAP were studied immunohistochemically on spinal cord tissue of 5 patients who died after unilateral infarction of the middle cerebral artery territory, and 5 control cases. In patients who died shortly after a stroke (4-14 days) increased HLA-DR-immunoreactivity (HLA-DR-IR) could be observed in the intermediate grey matter and in the ventral horn. The CD68-IR was much less intense. After longer survival times (5 weeks to 4 months). HLA-DR-IR in the grey matter was clearly lower than that observed in the spinal cord of short survival times, but very abundant in the dorsolateral funiculus, specifically within the corticospinal tract. In white matter areas, CD68-IR was almost identical to the HLA-DR-IR. Within the grey matter, CD68-IR was similar to the control tissue. A moderate increase of GFAP-positive astrocytes could be seen only in the grey matter after longer survival times. It seems probable, that the dynamics of HLA-DR-positive microglia reflect the early phagocytosis of presynaptic terminals by microglia in target regions of descending fibre tracts. In the white matter, the removal of degenerating axons by phagocytosing microglia expressing HLA-DR and CD68 antigens is a slower process which occurs over a period of months.     

Experimental studies of mechanically-induced articular cartilage defects following implantation of allogeneic embryonal chondrocytes in a collagen-fibrin gel in chickens

Full thickness defects (diameter 1,7 mm; depth 2,5 mm) were created mechanically in articular cartilage and subchondral bone of the condyles of tibiotarsal joints of 9-month old chickens. This full-thickness defects were repaired with cultured allogenic embryonic chick epiphyseal chondrocytes from the tibiae and femura of 10-days-old chicken embryos. The cells were embedded in a collagen-fibrinogen-matrix. Controls were similarly operated, but received either no treatment or implants the delivery substance only. Healing of the defects was observed macroscopically, histologically, histochemically and histomorphometrically after 3, 12 and 24 weeks. This graft was successfully transplanted in mechanically induced defects in 80%. The resulting hyaline cartilage was structurally reorganized according to the host pattern and under the influence of environmental conditions. The articular zone preserved it's cartilaginous phenotype, whereas the subchondral regions were transformed into bone. 12 weeks after the operation the defects in the experimental group were completely filled. In all instances in this group, there was an initial extreme increase of mitotic rate and cell number. After 24 weeks normal and subnormal values were founded. The defects in the control groups healed with fibrocartilage. Our results showed, that only the defects in the experimental group were completely filled with reparative hyaline cartilage tissue. In the present study the mixture of cultured allogenic embryonic chondrocytes and a collagen-fibrinogen-matrix was used successfully as a transplant for repairing defects in articular cartilage of chickens. Thus allogenic transplantation of cultured embryonal chondrocytes appears to be one of the most promising methods for the restoration of articular cartilage.     

Immunohistochemistry of articular cartilage from immature beagle dogs dosed with difloxacin

Effects of the fluoroquinolone difloxacin on articular-epiphyseal cartilage in growing beagle dogs have been described by light microscopic, electron microscopic, and biochemical methods. Here we present data from an immunohistochemistry study with humeral head cartilage from 3-mo-old beagle dogs after treatment with 1 or 2 oral doses of 300 mg difloxacin/kg body weight. Dogs were euthanatized either 24 hr (single dose) or 48 hr (2 doses) after onset of dosing, and cartilage tissue was stored at -90 degrees C until it was studied by immunohistochemistry. Antibodies against matrix components (collagen II, fibronectin) as well as antibodies against cellular structures (integrins) were used. After single-dose treatment (24-hr group), cartilage lesions such as clefts were not observed, but increased staining for fibronectin was found in cartilage samples from 5 of 6 animals. Markedly increased staining for fibronectin was also demonstrated in the vicinity of clefts within cartilage of all animals of the 48-hr group. Collagen II staining was homogeneously distributed in cartilage from controls and was slightly reduced in territorial matrix in 2 of 6 dogs of the 48-hr group. Integrin staining on chondrocytes was not significantly affected by difloxacin under the given conditions with the exception of a slight reduction of the alpha v integrin chain in 1 of 5 dogs of the 48-hr group. Overall, the most important results is the finding that fibronectin was a sensitive immunohistochemical marker for change in cartilage samples due to difloxacin treatment in dogs.     

Induction of release of secretory nonpancreatic phospholipase A2 from human articular chondrocytes

OBJECTIVE: Secretory nonpancreatic phospholipase A2 (sPLA2) is a known inducer/promoter of the inflammatory process in the joints. It correlates with disease activity in adult and juvenile rheumatoid arthritis. Synovial fluids contain high concentrations of sPLA2. We discovered that human articular cartilage contains large quantities of sPLA2 and that culture chondrocytes constitutively synthesize and release sPLA2. To test the mechanism controlling the release of sPLA2, we exposed cultured human articular chondrocytes to cytokines and other agents, known to induce sPLA2 in other cells. METHODS: Chondrocytes obtained from cartilage of normal appearance from rheumatoid and osteoarthritic joints, and from normal, neonatal joints were compared to rabbit articular chondrocytes. Radiolabeled Escherichia coli derived phospholipid assay and ELISA technique using monoclonal antibodies against recombinant human synovial type sPLA2 were employed. The cells were grown as monolayers as well as in alginate beads. RESULTS: Human articular chondrocytes from both arthritic and neonatal joints released sPLA2 constitutively but could not be further stimulated with interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha), IL-6, oncostatin M, lipopolysaccharide (LPS), or forskolin. Marked stimulation was observed when the cells were exposed to 8-bromo cyclic adenosine monophosphate (cAMP). Growing the cells as monolayers or in alginate beads did not change the results. In contrast to human cells, rabbit chondrocytes responded to IL-1 beta and IL-1/TNF, but not to TNF-alpha alone, with a very marked increase in extracellular sPLA2 activity. CONCLUSION: Human articular chondrocytes synthesize and constitutively release sPLA2. Such continuous release is most probably responsible for the high concentration of sPLA2 in articular cartilage and may be the source of synovial fluid sPLA2. To our knowledge, human articular chondrocytes are the only sPLA2 producing cells tested to date that do not respond to cytokine stimulation with increased sPLA2 activity; yet enhancement was seen with 8-bromo cAMP. It seems therefore that, human articular chondrocytes possess signalling mechanisms for the release of sPLA2 unlike those from other mammalian cells. The significance of this observation remains to be elucidated.     

Basic fibroblast growth factor and insulinlike growth factor I support the growth of human septal chondrocytes in a serum-free environment

OBJECTIVE: To determine if insulinlike growth factor I (IGF-I) and basic fibroblast growth factor (bFGF), individually or in combination, support the growth and viability of human septal chondrocytes in a serum-free medium (SFM) and a serum-enhanced culture medium. DESIGN: Chondrocytes were recovered from enzymatically digested human septal cartilage and were plated for monolayer culture in a newly developed medium. The medium included Dulbecco modified Eagle medium mixed 1:1 with Ham F12 medium and a supplement of known amounts of 2 growth factors-bFGF (100 ng/mL) and IGF-I (100 ng/mL)-used in combination and separately. RESULTS: The combination of IGF-I and bFGF enhanced chondrocyte growth and maintained a high degree of viability in SFM and 10% fetal calf serum. After an initial lag, the SFM, augmented with both growth factors, produced a comparable number of viable cells (4.25+/-0.31 x 10(4)) to that of the medium with 10% fetal calf serum (4.64+/-0.35 x 10(4)) by the seventh day of the experiment. Combined with the 2 growth factors, 10% fetal calf serum provided the greatest proliferation by the end of the experiment. However, the overall mean cell counts for the IGF-I- and bFGF-enhanced SFM were not statistically different. CONCLUSIONS: The combination of IGF-I and bFGF in a serum-free and a serum-supplemented environment supports the growth and viability of human septal chondrocytes in short-term culture. In an SFM, the results obtained approximate those produced in a medium enhanced with 10% fetal calf serum.     

Monoclonal antibody enhanced stimulation of human growth hormone action in vitro using ovine costal cartilage growth plate chondrocytes

Investigations into the mechanism underlying antibody-mediated enhancement of growth hormone action have been hampered by the lack of an in vitro assay system. In this work, we have used isolated ovine costal cartilage growth plate chondrocytes to demonstrate, for the first time, that monoclonal antibody EB1 can enhance the proliferative actions of human growth hormone on this cell type. Chondrocytes were cultured for 14 days prior to exposure to GH+/-monoclonal antibody EB1 for a 4-day treatment period. Human growth hormone alone promoted a significant dose-dependent increase in chondrocyte proliferation; maximal stimulation was achieved at about 3.3-10 ng/ml growth hormone, and at higher doses of growth hormone, the response declined. Monoclonal antibody EB1 was shown to enhance the proliferative activity of 10 ng/ml human growth hormone in a significant dose dependent fashion. In conclusion, our results demonstrate that an antibody capable of enhancing GH activity in vivo also has the capacity to potentiate GH activity in vitro. This system may provide an important tool for investigations into the mechanism of GH action, and how this is modified by GH enhancing MAbs.     

Parathyroid hormone-related proteins is abundant in osteoarthritic cartilage, and the parathyroid hormone-related protein 1-173 isoform is selectively induced by transforming growth factor beta in articular chondrocytes and suppresses generation of extracellular inorganic pyrophosphate

OBJECTIVE: Parathyroid hormone-related protein (PTHrP) is a major, locally expressed regulator of growth cartilage chondrocyte proliferation, differentiation, synthetic function, and mineralization. Because mechanisms that limit cartilage chondrocytes from maturing and mineralizing are diminished in osteoarthritis (OA), we studied PTHrP expression by articular chondrocytes. METHODS: PTHrP was studied in normal knee cartilage samples and cultured articular chondrocytes, and in cartilage specimens from knees with advanced OA, obtained at the time of joint replacement. RESULTS: PTHrP was more abundant in OA than in normal human knee articular cartilage. Both demonstrated PTH/PTHrP receptor expression. PTHrP 1-173, one of three alternatively spliced PTHrP isoforms, was exclusively expressed and induced by transforming growth factor beta in cultured chondrocytes. Chondrocytes mainly used the GC-rich P2 alternative promoter to express PTHrP messenger RNA. Inhibition by PTHrP 1-173, but not by PTHrP 1-146 or PTHrP 1-87, of inorganic pyrophosphate (PPi) elaboration suggested selective functional properties of the 1-173 isoform. Exposure to a neutralizing antibody to PTHrP increased PPi elaboration by articular chondrocytes. CONCLUSION: Increased expression of PTHrP, including the 1-173 isoform, has the potential to contribute to the pathologic differentiated functions of chondrocytes, including mineralization, in OA.     

Effects of fibronectin on articular cartilage chondrocyte proteoglycan synthesis and response to insulin-like growth factor-I

CD30 engagement in human lymphoid cells induces pleiotropic cellular responses that affect cellular viability and proliferation, cytokine production, and nuclear factor kappaB (NF-kappaB) nuclear translocation. Studies examining the molecular basis for this pleiotropism thus far have relied on the use of antibodies and cells transfected with CD30L to trigger CD30, two methods of receptor induction that present important limitations: antibodies are not physiological receptor-triggering molecules and CD30L transfectants induce high background intracellular signaling in the cells under study. We have generated and expressed a functional soluble human CD30L molecule (sCD30L/CD8alpha) comprised of the extracellular domain of human CD30L fused to the extracellular domain of the human CD8alpha chain. Immunoprecipitation and Western blot analysis of sCD30L/CD8alpha revealed the existence of at least two forms of sCD30L/CD8alpha, which exhibited molecular sizes consistent with the existence of monomeric and trimeric forms of the molecule. Binding analyses performed using a soluble CD30 fusion protein (sCD30/gamma1) confirmed the ability of sCD30L/CD8alpha to bind to CD30. Functionally, immobilized sCD30L/CD8alpha-induced cell death in the CD30-expressing lines Karpas-299 and HDLM-2 and reduced proliferative levels in Karpas-299; these effects were inhibitable by the addition of sCD30/gamma1. These studies demonstrate the utility of sCD30L/CD8alpha in characterizing the normal function of CD30L and CD30 and indicate the natural ability of soluble forms of CD30L to trimerize.