Toward understanding the role of cartilage particulates in synovial inflammation.

OBJECTIVE: Arthroscopy with lavage and synovectomy can remove tissue debris from the joint space and the synovial lining to provide pain relief to patients with osteoarthritis (OA). Here, we developed an in vitro model to study the interaction of cartilage wear particles with fibroblast-like synoviocytes (FLS) to better understand the interplay of cartilage particulates with cytokines on cells of the synovium. METHOD: In this study sub-10 µm cartilage particles or 1 µm latex particles were co-cultured with FLS ±10 ng/mL interleukin-1α (IL-1α) or tumor necrosis factor-α (TNF-α). Samples were analyzed for DNA, glycosaminoglycan (GAG), and collagen, and media samples were analyzed for media GAG, nitric oxide (NO) and prostaglandin-E2 (PGE2). The nature of the physical interaction between the particles and FLS was determined by microscopy. RESULTS: Both latex and cartilage particles could be phagocytosed by FLS. Cartilage particles were internalized and attached to the surface of both dense monolayers and individual cells. Co-culture of FLS with cartilage particulates resulted in a significant increase in cell sheet DNA and collagen content as well as NO and PGE2 synthesis compared to control and latex treated groups. CONCLUSION: The proliferative response of FLS to cartilage wear particles resulted in an overall increase in extracellular matrix (ECM) content, analogous to the thickening of the synovial lining observed in OA patients. Understanding how cartilage particles interface with the synovium may provide insight into how this interaction contributes to OA progression and may guide the role of lavage and synovectomy for degenerative disease.

The lymphocyte in immunology: from James B. Murphy to James L. Gowans.

Between 1912 and 1921, James Murphy established conclusively the role of the lymphocyte in tissue and tumor graft rejection and in protection against infection. Contemporary mainstream immunology paid little attention to these findings, until the lymphocyte was "rediscovered" with the advent of modern cellular immunology after the mid-1950s.

There is only one immune system! The view from immunopathology.

The generators of B and T cell diversity produce specificities for both autochthonous and exogenous paratopes. A wide variety of positive and negative, central and peripheral mechanisms has evolved to regulate the immune response. All potential immunogens are recognized by the system using the same set of 'rules', without discrimination between 'self' and 'nonself' or between the 'toxic' and the 'benign'. In every response, whether positive or negative, the factors mobilized and the balance between protection and damage depend upon the quality, quantity, location, and timing of immunogen presentation, as well as upon properties of the host.

The Hsp organizer protein hop enhances the rate of but is not essential for glucocorticoid receptor folding by the multiprotein Hsp90-based chaperone system.

A system consisting of five purified proteins: Hsp90, Hsp70, Hop, Hsp40, and p23, acts as a machinery for assembly of glucocorticoid receptor (GR).Hsp90 heterocomplexes. Hop binds independently to Hsp90 and to Hsp70 to form a Hsp90.Hop.Hsp70.Hsp40 complex that is sufficient to convert the GR to its steroid binding form, and this four-protein complex will form stable GR.Hsp90 heterocomplexes if p23 is added to the system (Dittmar, K. D., Banach, M., Galigniana, M. D., and Pratt, W. B. (1998) J. Biol. Chem. 273, 7358-7366). Hop has been considered essential for the formation of receptor.Hsp90 heterocomplexes and GR folding. Here we use Hsp90 and Hsp70 purified free of all traces of Hop and Hsp40 to show that Hop is not required for GR.Hsp90 heterocomplex assembly and activation of steroid binding activity. Rather, Hop enhances the rate of the process. We also show that Hsp40 is not essential for GR folding by the five-protein system but enhances a process that occurs less effectively when it is not present. By carrying out assembly in the presence of radiolabeled steroid to bind to the GR as soon as it is converted to the steroid binding state, we show that the folding change is brought about by only two essential components, Hsp90 and Hsp70, and that Hop, Hsp40, and p23 act as nonessential co-chaperones.

Pasteur, Pastorians, and the dawn of immunology: the importance of specificity.

Throughout his career, the problems that attracted Louis Pasteur almost invariably involved considerations of specificity of structure and/or of action. Thus, his work on asymmetric crystals showed that chemical form not only specifies crystalline structure, but affects the affinity of ferments as well. In his studies of diseases of silkworms, of beer, and of wine, he could unerringly distinguish with the microscope the specific agents of disease. From this emerged his concept of the specificity of species and against the nonspecificity of spontaneous generation, whence the germ theory of disease. It was in the new field of immunology, however, where the manifestations of an exquisite specificity were most clearly seen. Here, Pasteur's vaccines worked because he chose the specific pathogen in order to induce a specific immunity, and he succeeded each time. But the two most prominent Pastorian successors in immunology, Elie Metchnikoff and Jules Bordet, were not equally successful. Although each contributed significantly to the birth of immunology, each advanced a theory that neglected the principle of specificity and paid a price in consequence. Metchnikoff's phagocytic theory of immunity could not survive the demonstrable specificity of humoral antibodies, while Bordet's physical adsorptive concept of the antibody-cell interaction quickly fell to Paul Ehrlich's demonstration of the stereochemical determination of immunological specificity.

The most elegant immunological experiment of the XIX century.

When we remember that immunology was barely a decade old and knowledge of circulating antibodies only two years old when Ehrlich performed the experiments described, we can appreciate the inventiveness of his experimental designs. With little wasted effort he planned simple and rapid experiments to answer crucial questions about the mechanism of passive transfer of antibody from mother to fetus to suckling young. Most remarkable and difficult were the foster mother experiments, as anyone who has tried these with mice will attest. But they were judged to be critical and thus were pursued with ultimate success. These experiments would not be improved upon for 60 years, when the identification of immunoglobulin classes made differential transplacental and transglandular passage of immunoglobulins an object of interest. To conceive of studying the kinetics of the immune response by measuring changes in antibody concentration in the milk of lactating animals was yet another demonstration of the fertile imagination that had contributed so much to histology and hematology, and would soon contribute equally to experimental oncology and to scientific pharmacology.

The seven amino acids (547-553) of rat glucocorticoid receptor required for steroid and hsp90 binding contain a functionally independent LXXLL motif that is critical for steroid binding.

Hsp90 association with glucocorticoid receptors (GRs) is required for steroid binding. We recently reported that seven amino acids (547-553) overlapping the amino-terminal end of the rat GR ligand-binding domain are necessary for hsp90 binding, and consequently steroid binding. The role of a LXXLL motif at the COOH terminus of this sequence has now been analyzed by determining the properties of Leu to Ser mutations in full-length GR and glutathione S-transferase chimeras. Surprisingly, these mutations decreased steroid binding capacity without altering receptor levels, steroid binding affinity, or hsp90 binding. Single mutations in the context of the full-length receptor did not affect the transcriptional activity but the double mutant (L550S/L553S) was virtually inactive. This biological inactivity was found to be due to an increased rate of steroid dissociation from the activated mutant complex. These results, coupled with those from trypsin digestion studies, suggest a model in which the GR ligand-binding domain is viewed as having a "hinged pocket," with the hinge being in the region of the trypsin digestion site at Arg(651). The pocket would normally be kept shut via the intramolecular interactions of the LXXLL motif at amino acids 550-554 acting as a hydrophobic clasp.

Different regions of the immunophilin FKBP52 determine its association with the glucocorticoid receptor, hsp90, and cytoplasmic dynein.

FKBP52 is a high molecular mass immunophilin possessing peptidylprolyl isomerase (PPIase) activity that is inhibited by the immunosuppressant drug FK506. FKBP52 is a component of steroid receptor.hsp90 heterocomplexes, and it binds to hsp90 via a region containing three tetratricopeptide repeats (TPRs). Here we demonstrate by cross-linking of the purified proteins that there is one binding site for FKBP52/dimer of hsp90. This accounts for the common heterotetrameric structure of native receptor heterocomplexes being 1 molecule of receptor, 2 molecules of hsp90, and 1 molecule of a TPR domain protein. Immunoadsorption of FKBP52 from reticulocyte lysate also yields co-immunoadsorption of cytoplasmic dynein, and we show that co-immunoadsorption of dynein is competed by a fragment of FKBP52 containing its PPIase domain, but not by a TPR domain fragment that blocks FKBP52 binding to hsp90. Using purified proteins, we also show that FKBP52 binds directly to the hsp90-free glucocorticoid receptor. Because neither the PPIase fragment nor the TPR fragment affects the binding of FKBP52 to the glucocorticoid receptor under conditions in which they block FKBP52 binding to dynein or hsp90, respectively, different regions of FKBP52 must determine its association with these three proteins.

Glucocorticoids stimulate the accumulation of lipids in the rat corpus luteum.

We investigated the physiological basis for the trophic effect of glucocorticoids in rat corpora lutea in the absence of pituitary gonadotropins. Immature (Day 29) Sprague-Dawley rats were given eCG and hCG to induce the development of corpora lutea and were hypophysectomized on Day 32. Beginning on Day 40, rats received twice-daily s.c. injections of either dexamethasone (dex; 200 microg/rat/day) or vehicle (controls) and then were killed on Day 44. Plasma 20alpha-dihydroprogesterone, a major steroid produced by the corpora lutea, was higher (p 2-fold of plasma 20alpha-dihydroprogesterone concentration compared to controls. Glucocorticoid receptor protein (about 92 kDa) was detected in both luteal and nonluteal ovarian tissues in this animal model. These effects of glucocorticoids and the presence of the glucocorticoid receptor raise the possibility of a physiological role for glucocorticoids in the rat corpus luteum.

Paul Ehrlich's passion: the origins of his receptor immunology.

In 1897, Paul Ehrlich published a selection theory of antibody formation that anticipated the theories of Jerne and Burnet by some 60 years. Ehrlich introduced into immunology the concept of the interaction of physiologically active substances with specific receptors, an idea that still dominates modern immunological thought. In this paper, we point out how Ehrlich's concept matured over 20 years, while it governed his studies in histological staining, in cell physiology, in hematology, and finally in his major contributions in experimental immunology.

Neuronal nitric-oxide synthase is regulated by the Hsp90-based chaperone system in vivo.

It is established that the multiprotein heat shock protein 90 (hsp90)-based chaperone system acts on the ligand binding domain of the glucocorticoid receptor (GR) to form a GR.hsp90 heterocomplex and to convert the receptor ligand binding domain to the steroid-binding state. Treatment of cells with the hsp90 inhibitor geldanamycin inactivates steroid binding activity and increases the rate of GR turnover. We show here that a portion of neuronal nitric-oxide synthase (nNOS) exists as a molybdate-stabilized nNOS. hsp90 heterocomplex in the cytosolic fraction of human embryonic kidney 293 cells stably transfected with rat nNOS. Treatment of human embryonic kidney 293 cells with geldanamycin both decreases nNOS catalytic activity and increases the rate of nNOS turnover. Similarly, geldanamycin treatment of nNOS-expressing Sf9 cells partially inhibits nNOS activation by exogenous heme. Like the GR, purified heme-free apo-nNOS is activated by the DE52-retained fraction of rabbit reticulocyte lysate, which also assembles nNOS. hsp90 heterocomplexes. However, in contrast to the GR, heterocomplex assembly with hsp90 is not required for increased heme binding and nNOS activation in this cell-free system. We propose that, in vivo, where access by free heme is limited, the complete hsp90-based chaperone machinery is required for sustained opening of the heme binding cleft and nNOS activation, but in the heme-containing cell-free nNOS-activating system transient opening of the heme binding cleft without hsp90 is sufficient to facilitate heme binding.

A model for the cytoplasmic trafficking of signalling proteins involving the hsp90-binding immunophilins and p50cdc37.

A number of transcription factors and protein kinases involved in signal transduction exist in heterocomplexes with the ubiquitous and essential protein chaperone hsp90. These signalling protein x hsp90 heterocomplexes are assembled by a multiprotein chaperone system comprising hsp90, hsp70, Hop, hsp40, and p23. In the case of transcription factors, the heterocomplexes with hsp90 also contain a high molecular weight immunophilin with tetratricopeptide repeat (TPR) motifs, such as FKBP52 or CyP-40. In the case of the protein kinases, the heterocomplexes contain p50cdc37. The immunophilins bind to a single TPR acceptor site on hsp90, and p50cdc37 binds to an adjacent site so that binding is exclusive for p50cdc37 or an immunophilin. Direct interaction of immunophilins with the transcription factors or p50cdc37 with the protein kinases leads to selection of different heterocomplexes after their assembly by a common mechanism. Studies with the glucocorticoid receptor, for which translocation from the cytoplasm to the nucleus is under hormonal control, suggest that dynamic assembly of the heterocomplexes is required for rapid movement of the receptor through the cytoplasm along cytoskeletal tracts. As for the similar short-range trafficking of vesicles along microtubules, there must be a mechanism for linking the signalling protein solutes to the molecular motors involved in movement. We present here a model in which the immunophilins and p50cdc37 target, respectively, the retrograde or anterograde direction of signalling protein movement by functioning as connectors that link the signalling proteins to the movement machinery.

High-molecular-weight FK506-binding proteins are components of heat-shock protein 90 heterocomplexes in wheat germ lysate.

In animal cell lysates the multiprotein heat-shock protein 90 (hsp90)-based chaperone complexes consist of hsp70, hsp40, and p60. These complexes act to convert steroid hormone receptors to their steroid-binding state by assembling them into heterocomplexes with hsp90, p23, and one of several immunophilins. Wheat germ lysate also contains a hsp90-based chaperone system that can assemble the glucocorticoid receptor into a functional heterocomplex with hsp90. However, only two components of the heterocomplex-assembly system, hsp90 and hsp70, have thus far been identified. Recently, purified mammalian p23 preadsorbed with JJ3 antibody-protein A-Sepharose pellets was used to isolate a mammalian p23-wheat hsp90 heterocomplex from wheat germ lysate (J.K. Owens-Grillo, L.F. Stancato, K. Hoffmann, W.B. Pratt, and P. Krishna [1996] Biochemistry 35: 15249-15255). This heterocomplex was found to contain an immunophilin(s) of the FK506-binding class, as judged by binding of the radiolabeled immunosuppressant drug [3H]FK506 to the immune pellets in a specific manner. In the present study we identified the immunophilin components of this heterocomplex as FKBP73 and FKBP77, the two recently described high-molecular-weight FKBPs of wheat. In addition, we present evidence that the two FKBPs bind hsp90 via tetratricopeptide repeat domains. Our results demonstrate that binding of immunophilins to hsp90 via tetratricopeptide repeat domains is a conserved protein interaction in plants. Conservation of this protein-to-protein interaction in both plant and animal cells suggests that it is important for the biological action of the high-molecular-weight immunophilins.

Acute postictal cerebral imaging.

BACKGROUND AND PURPOSE: Imaging of postictal patients is performed to investigate causes of seizure, such as space-occupying lesions or other "structural" processes; however, abnormalities may be found that reflect physiological or pathologic alterations due to seizure activity. The purpose of this study was to determine the brain imaging findings in patients in the immediate postictal period who presented with altered mental status or weakness. METHODS: Ten patients who were examined for postictal neurologic derangement were studied (nine by CT and one by MR imaging) within 12 hours of ictus. Four of the CT studies and the one MR study included administration of contrast material. Follow-up examinations were performed 1 day to 11 months later. These studies were reviewed retrospectively. RESULTS: CT findings included focal gyral swelling (10/10), effacement of adjacent cortical sulci (2/10), decreased gyral attenuation by CT (8/9), and mild to moderate gyral enhancement after injection of contrast material (5/5). MR imaging findings included gyral swelling, increased signal intensity on T2-weighted images, and enhancement after injection of contrast agent. The abnormalities were located in the frontal lobes (9/10, with bilateral involvement in 6/10), the parietal lobes (4/10), the temporal lobes (2/10), and the occipital lobe (1/10). Follow-up studies revealed complete or subtotal reversal of these abnormalities. CONCLUSION: Although there are numerous causes of gyral swelling and enhancement, such as infarction and neoplasm, if these conditions are reversible and correspond to clinical findings, then the differential diagnosis is narrowed to postictal change, reversible ischemia, complicated migraine, or resolved inflammation/infection.

p50(cdc37) binds directly to the catalytic domain of Raf as well as to a site on hsp90 that is topologically adjacent to the tetratricopeptide repeat binding site.

Several protein kinases (e.g. pp60(src), v-Raf) exist in heterocomplexes with hsp90 and a 50-kDa protein that is the mammalian homolog of the yeast cell cycle control protein Cdc37. In contrast, unliganded steroid receptors exist in heterocomplexes with hsp90 and a tetratricopeptide repeat (TPR) domain protein, such as an immunophilin. Although p50(cdc37) and TPR domain proteins bind directly to hsp90, p50(cdc37) is not present in native steroid receptor.hsp90 heterocomplexes. To obtain some insight as to how v-Raf selects predominantly hsp90.p50(cdc37) heterocomplexes, rather than hsp90.TPR protein heterocomplexes, we have examined the binding of p50(cdc37) to hsp90 and to Raf. We show that p50(cdc37) exists in separate hsp90 heterocomplexes from the TPR domain proteins and that intact TPR proteins compete for p50(cdc37) binding to hsp90 but a protein fragment containing a TPR domain does not. This suggests that the binding site for p50(cdc37) lies topologically adjacent to the TPR acceptor site on the surface of hsp90. Also, we show that p50(cdc37) binds directly to v-Raf, with the catalytic domain of Raf being sufficient. We propose that the combination of exclusive binding of p50(cdc37) versus a TPR domain protein to hsp90 plus direct binding of p50(cdc37) to Raf allows the protein kinase to select for the dominant hsp90.p50(cdc37) composition that is observed with a variety of protein kinase heterocomplexes immunoadsorbed from cytosols.