Steven M. Friedman

Characterization of cartilage metabolic response to static and dynamic stress using a mechanical explant test system

A new mechanical explant test system was used to study the metabolic response (via proteoglycan biosynthesis) of mature, weight-bearing canine articular cartilage subjected to static and dynamic compressive stresses. Stresses ranging from 0.5 to 24 MPa were applied sinusoidally at 1 Hz for intervals of 2-24 h. The explants were loaded in unconfined compression and compared to age-matched unloaded explants. Both static and dynamic compressive stress significantly decreased proteoglycan biosynthesis (range 25-85%) for all loading time intervals. The inhibition was proportional to the applied stress but was independent of loading time. After rehydration upon load removal, the measured water content of the loaded explants was not different from the unloaded explants for all test variables. Autoradiographic and electron microscopic analysis of loaded explants showed viable chondrocytes throughout the matrix. Our results suggest that the decreased metabolic response of cyclically loaded explants may be dominated by the static component (RMS) of the dynamic load. Furthermore, the observed decreased metabolism may be more representative of the in situ tissue response than that of unloaded explants, in which we found an increasing rate of metabolism for up to 6 days after explant removal.

CDR3 sequence motifs shared by oligoclonal rheumatoid arthritis synovial T cells. Evidence for an antigen-driven response.

T lymphocytes reactive with as yet undefined joint-localized foreign or autoantigens may be important in the pathogenesis of RA. Molecular studies demonstrating skewed T cell antigen receptor (TCR) variable gene usage and selective expansion of particular T cell clones within the synovial compartment support this view. Based on our recent study documenting selective expansion of V beta 17+ T cells in RA, we have pursued the identification of T cells relevant to the disease process, in an informative patient, by combining molecular analysis of freshly explanted RA synovial tissue V beta 17 TCR transcripts with in vitro expansion of V beta 17+ synovial tissue T cell clones. Peripheral blood V beta 17 cDNA transcripts proved heterogeneous. In contrast, two closely related sequences, not found in the peripheral blood, dominated synovial tissue V beta 17 transcripts, suggesting selective localization and oligoclonal expansion at the site of pathology. CD4+, V beta 17+ synovial tissue-derived T cell clones, isolated and grown in vitro, were found to express TCR beta chain transcripts homologous to the dominant V beta 17 synovial tissue sequences. One clone shares with a dominant synovial tissue sequence a conserved cluster of 4/5 amino acids (IGQ-N) in the highly diverse antigen binding CDR3 region, suggesting that the T cells from which these transcripts derive may recognize the same antigen. These findings have permitted a complete characterization of the alpha/beta TCR expressed by putatively pathogenic T cell clones in RA. Functional analysis suggests that the conserved CDR3 sequence may confer specificity for, or restriction by, the MHC class II antigen, DR4.

Fas expression and apoptosis in human B cells

Mechanisms of B cell apoptosis are critical in reducing aberrant B cell proliferations such as those that arise in autoimmune disease and in B cell malignancies. The physiologic interaction of CD4+ helper T cells and B lymphocytes has been extensively studied over the past two decades. Although CD4+ T cells are considered primarily to offer positive costimulatory signals for B cell differentiation into active immunoglobulin-secreting cells, recent studies have shown that CD4+ T cells are crucial in downregulating the humoral immune response. In the course of cognate interaction between CD40 ligand (CD40L)-bearing CD4+ T cells and CD40-expressing germinal center B cells, CD40 ligation results in augmented Fas expression at the B cell surface. Like CD40L, Fas ligand is expressed on activated CD4+ Th1 cells and when bound to Fas receptor on the B cell surface, initiates an apoptotic signal in that cell. Thus, CD4+ T cells limit the growth of autologous germinal center B cells by first inducing Fas expression and then instigating a death signal via Fas ligand. In this work, we will consider these observations about CD4+ T-cell-induced, Fas-mediated B cell death in the context of other factors that affect apoptosis in B cells, normal and malignant.

Human B cell differentiation induced by microbial superantigens : unselected peripheral blood lymphocytes secrete polyclonal immunoglobulin in response to Mycoplasma arthritidis mitogen

Microbial superantigens (SA) activate a significant portion of the T cell repertoire based on their dual avidity for MHC class II antigens and T cell receptor (TCR) epitopes common to products of one or several TCR beta chain variable gene families. While SA that induce massive T cell proliferation and cytokine secretion have been implicated in clinical syndromes characterized by shock and generalized immunosuppression, SA activation of a more restricted T cell response may also have significant, perhaps immunostimulatory, effects on the immune system. To investigate this issue, we measured 3H-thymidine incorporation and polyclonal IgM and IgG secretion by normal human peripheral blood mononuclear cells (PBMC) cultured with a panel of microbial SA, including the Staphylococcus aureus-derived SA, SEA, SEB, SEC-1, SEC-2, SEC-3, SEE, TSST-1, and the Mycoplasma arthritidis-derived SA, MAM. The S. aureus-derived SA induce vigorous proliferation by PBMC, while optimal MAM-induced proliferation is significantly lower in magnitude. In all 12 subjects tested, mitogenic concentrations of MAM reproducibly stimulate unselected PBMC to secrete polyclonal IgM and IgG. In contrast, the S. aureus-derived SA induce Ig production only in cultures containing isolated B cell populations and either very low numbers of untreated autologous T cells, larger numbers of X-irradiated autologous T cells, or very low concentrations of the SA. No difference in the activation of helper (CD4) versus suppressor/cytotoxic (CD8) T cells by MAM and the S. aureus-derived SA was noted. Taken together, these data suggest that MAMs capacity to induce B cell differentiation correlates with its induction of a relatively weak proliferative response by unselected human T cells. MAM-like SA, when encountered in vivo, may result in a significant perturbation of the human immune system and potentially contribute to clinical syndromes characterized by immunostimulation and hypergammaglobulinemia.

Human immunodeficiency virus infection of helper T cell clones. Early proliferative defects despite intact antigen-specific recognition and interleukin 4 secretion.

HIV selectively inhibited the proliferative response of clonal CD4+ T lymphocytes to alloantigen while other alloantigen-dependent responses were unperturbed. Specifically, impaired blastogenesis could be dissociated from alloantigen-specific induction of the B cell activation molecule CD23, IL-4 release, and inositol lipid hydrolysis. In addition, membrane expression of pertinent T cell receptor molecules, including CD2, CD3, and T cell antigen receptor (Ti), remained intact. Using two MHC class II-specific human CD4+ helper T cell clones, the proliferative defect was shown to be an early consequence of HIV infection, occurring within 4 d of viral inoculation and preceding increases in mature virion production. It was generalizable to three distinct methods of T cell activation, all independent of antigen-presenting cells: anti-CD3 mediated cross-linking of the CD3/Ti complex; anti-CD2 and phorbol 12-myristic 13-acetate (PMA); and anti-CD28 plus PMA. These abnormalities were not mitigated by addition of exogenous IL-2, even though expression of the IL-2 receptor (CD25) was unaltered. These studies define a selective blockade in T cell function early after HIV exposure that could serve as a model for certain in vivo manifestations of AIDS.

Human peripheral blood T helper cell-induced B cell activation results in B cell surface expression of the CD23 (BLAST-2) antigen

We have developed an in vitro system to assess the early stages of B cell activation induced by peripheral blood T helper cells. Peripheral blood mononuclear cells are cultured for 16 hr with anti-CD3 monoclonal antibody (mAb), T lymphocytes are then removed by sheep red blood cell rosette depletion, and expression of the B cell surface activation antigen CD23 (BLAST-2) is assessed by indirect immunofluorescence. Anti-CD3 mAb, but not a control anti-CD5 mAb, stimulates the expression of CD23 on 20-50% of peripheral blood B cells cultured with autologous T cells. T cell subset depletion studies show that the CD4+ T cell subset is responsible for anti-CD3-mediated induction of CD23 on autologous B cells. Anti-CD3-induced, T helper cell-dependent CD23 expression is not MHC-restricted, as allogeneic combinations of T and non-T cells, cultured in the presence of anti-CD3 antibody, also result in the expression of B cell CD23. Individuals whose monocyte Fc receptors bind murine IgG1 mAb poorly fail to trigger T cell proliferation in response to murine IgG1 anti-CD3 mAb and also fail to express B cell CD23 following culture of PBMC with IgG1 anti-CD3 mAb, while the usual expression of CD23 is seen after culture with IgG2a anti-CD3 mAb. The mechanism of anti-CD3-induced B cell activation was addressed in experiments using a two-chamber culture system. While little IL-4 activity was detected in anti-CD3-stimulated culture supernatants, optimal induction of CD23 was observed when T and B cells were cultured together in a single chamber. This suggests that under physiologic conditions, in which quantities of lymphokine may be limiting, close physical contact between the anti-CD3-activated Th cell and B cell may be required for CD23 expression. The anti-CD3-induced BLAST-2 assay will facilitate the analysis of Th cell-mediated B cell activation in any individual and should permit us to separately evaluate the roles of Th cells and B cells in the impaired immunoregulation characteristic of autoimmune disorders.

T helper cell-dependent, microbial superantigen-mediated B cell activation in vivo

We have utilized a severe combined immune-deficient (SCID) mouse adoptive transfer model to explore the in vivo immunostimulatory effects of bacterial superantigens (SAg). B cell reconstituted SCID recipients were treated with the Staphylococcus aureus-derived toxic shock syndrome toxin (TSST-1) alone or in conjunction with syngeneic L3T4+ TSST-1-reactive Th cells. Over several months of study, the repetitive administration of TSST-1 resulted in a prompt, transient increase in serum IgG levels. This response required both biologically active TSST-1 and Th cells. These findings demonstrate that certain bacterial SAgs can promote Th cell-dependent B cell activation and differentiation in vivo. These studies strengthen the analogy between SAg-mediated and allospecific Th-B cell interactions responsible for the autoimmune sequelae of graft-versus-host disease.

CD40 ligation impedes lymphoblastoid B cell proliferation and S-phase entry

The CD40 cell surface antigen and member of the tumor necrosis factor (TNF) receptor superfamily is expressed in many cell types, including normal and neoplastic B cells. Signaling through CD40 induces B cell proliferation, differentiation and, in some circumstances, protects the B cell from apoptosis. Lymphoblastoid cells (LCLs) resemble the malignant B cells that comprise the Epstein-Barr virus (EBV)-associated posttransplant lymphoproliferative disorders, in that the cells bear a highly activated phenotype and, unlike most other EBV positive tumor cells, express the majority of latent EBV genes. In this study, we use assays of cell viability, proliferation, cell cycle and apoptosis to demonstrate that ligation of the CD40 receptor in EBV-transformed LCLs inhibits their growth. The process does not involve apoptosis, but is characterized by reduced S-phase entry from G0/G1. A better understanding of the negative effects of CD40 ligation in these cells may offer clues for the development of novel therapies in EBV-related B cell disorders.

Allelic variants of human TCR BV17S1 defined by restriction fragment length polymorphism, single strand conformation polymorphism, and amplification refractory mutation system analyses

Several human TCR BV gene subfamilies, including BV3, BV14, and BV17S1, are single member genes but are overutilized among activated CD4+ synovial T cells in the rheumatoid arthritis (RA). To define the role of these TCR BV genes in the pathogenesis of disease, it is critical to characterize the genomic organization and the allelic variations of these genes. In this study we describe allelic variations of BV17S1 defined by restriction fragment length polymorphism (RFLP), single strand conformation polymorphism (SSCP), and amplification refractory mutation system (ARMS) analyses. A single nucleotide replacement (C/T) results in an amino acid substitution (F/L) in the leader and distinguishes BV17S1*1 from BV17S1*2. This nucleotide substitution was found to create a BsmAI restriction enzyme recognition site in BV17S1*2. Therefore genotypic analyses can be performed either by the SSCP or RFLP method. The analyses of 75 unrelated individuals show that the frequency for allele BV17S1*1 is 52.7% and for allele BV17S1*2 is 47.3%. Both alleles are functionally expressed and are distributed within CD4+/CD8+ T cell subsets. Another point mutation in the CDR2 region of BV17S1, which results in the amino acid replacement of Gln by His, originally identified form a cDNA clone, has now been confirmed as an allele by ARMS analysis using genomic DNA preparations and designated to as BV17S1*3. Screening of this CDR2 related variant among normal populations indicates that this is a rare allele (1 of 75). Although this variant may be of functional significance, the genotypic analysis and functional studies are difficult due to the low frequency of BV17S1*3. In an attempt to define a correlation between BV17S1 allelic usage and susceptibility to RA, the germline distribution of BV17S1 alleles *1 and *2 has been examined in a small number of RA patients and no skewed usage has been identified.

Biochemical basis of synergy between antigen and T-helper (Th) cell-mediated activation of resting human B cells.

We have utilized CD23 expression as a marker for B cell activation in order to investigate the biochemical basis for synergy between antigen and T helper (Th) cells in the activation of resting human B cells. Our results confirm that while ligation of surface immunoglobulin (sIg) receptors by antigen analogues (e.g., F(ab)2 goat anti-human IgM) does not lead to CD23 expression, this stimulus markedly enhances CD23 expression induced during antigen specific Th-B cell interaction or by rIL-4. Utilizing a panel of monoclonal anti-human IgM antibodies, we observed a positive correlation between the capacity of a particular antibody to synergize with rIL-4 in CD23 expression and with B cell growth factor in B cell proliferation; suggesting that synergy in CD23 expression reflects the transduction of a functionally important signal via the sIg receptor. We next assayed analogues of the second messenger molecules, released during inositol lipid hydrolysis, for their capacity to amplify CD23 expression. These studies showed that protein kinase C (PKC) activating phorbol esters and the synthetic diacylgylcerol analogue, DiC8, synergize with either Th cells or rIL-4 in CD23 expression, while under no experimental condition does increasing B cell [Ca2+]i with ionomycin enhance CD23 expression. Taken together, these data suggest that activation of B cell PKC is the crucial biochemical event that primes antigen-activated B cells to respond more vigorously to interaction with Th cells and/or their soluble products.