Patients with rheumatoid arthritis have a subset of CD4+ T lymphocytes that are characterized by a defect in CD28 expression. CD4+CD28- T cells frequently undergo clonal expansion in vivo. These ...clonotypes include autoreactive cells and persist over many years. The clonogenic potential and longevity of these T cells could be related to an altered response to apoptosis-inducing signals. To explore this possibility, CD4+CD28- T cell lines and clones were examined for their response pattern to stimuli inducing physiologic cell death. CD4+CD28- T cells were found to be resistant to apoptosis upon withdrawal of the growth factor, IL-2. To examine whether the altered sensitivity to this apoptotic signal was correlated with the expression of proteins of the bcl-2 family, the expression of bcl-2, bcl-x, and bax proteins was determined. CD28+ and CD28-CD4+ T cells could not be distinguished by the levels of bax or bcl-xL protein; however, CD4+CD28- T cells expressed higher amounts of bcl-2 protein than did CD4+CD28+ T cells. The increased bcl-2 expression in CD4+CD28- T cells was relatively independent of signals provided by exogenous IL-2. In CD28-deficient CD4+ T cells, bcl-2 was not significantly up-regulated by the addition of exogenous IL-2 and was maintained despite IL-2 withdrawal, as opposed to CD28-expressing CD4+ T cells. We propose that CD4+CD28- T cells are characterized by a dysregulation of the survival protein, bcl-2, which may favor the clonal outgrowth of autoreactive T cells and thus contribute to the pathogenesis of rheumatoid arthritis.
Abstract only
INTRODUCTION
Past research in disc biology has highlighted the beneficial effects of growth factors, such as BMPs and IGF‐1,
in vitro
. Based on this research, it was proposed that ...exogenous IGF‐1 be used as a therapy for intervertebral disc degeneration (IDD). In the aging research field, suppressing the IGF‐1‐mediated signaling pathway has consistently decreased the incidence of age‐related disorders and increased lifespan of mammals and rodents. One model using
PappA
knockout mice has shown decreasing IGF‐1 signaling leads to positive effects on aging and longevity. Active PappA protease cleaves IGF binding protein 4 (IGFBP4), releasing active IGF‐1 which can bind to its receptor and exert downstream signaling effects. When you genetically remove PappA, IGF‐1 stays bound to IGFBP4. Here, we examined the discs of aged
PAPPA−/−
mice versus wild type (WT) to determine whether there was an effect on age‐associated IDD.
METHODS
This study was conducted according to IACUC approved protocols at University of Pittsburgh. Genetic deletion of PAPP‐A protease in mice with a mixed C57BL/6‐129SV/E background was achieved by deletion of both alleles of PAPP‐A. Spine segments were harvested from
WT
and
PAPPA−
/
−
mice at 23 months. Disc proteoglycan content was assessed by safranin‐O‐histology and DMMB assay was used to quantify sulfated glycosaminoglycans (GAG) in lumbar NP tissue. Western blotting was performed to measure the disc aggrecan proteolytic fragments, and the catabolic markers MMP‐3 and ADAMTS‐4. Levels of disc cellular senescence (loss of lamin B1expression) was assessed by immunohistochemistry. Student’s t‐test was used to test significance between groups (p<0.05). Average values (n=4 mice) were shown with SEM.
RESULTS
Compared to WT,
PAPPA−
/
−
mice had decreased proteoglycan content and two times less disc GAG content (Figure
1
).
PAPPA−
/
−
mice also had significantly decreased aggrecan fragmentation and decreased protein levels of MMP‐3 and ADAMTS‐4 in their discs compared to WT mice (Figure
2
). Additionally, greater level of disc lamin B1 was seen in
PAPPA
−/− compared to WT mice, suggesting reduced disc cellular senescence in
PAPPA
−/− mice.
DISCUSSION
The role of IGF‐1 in disc health and aging is currently unresolved.
PAPPA
−/− mice have reduced matrix content but the aggrecan matrix present after aging appears to be less fragmented based on our findings. Lowered disc fragmentation corresponded to a decrease in disc catabolic factors MMP‐3 and ADAMTS‐4 protein in
PAPPA
−/− mice. Lamin B1 staining suggests there are less disc senescent cells in old
PAPPA
−/− mice compared to normally aged mice. Further studies are needed to determine the mechanism for the decreased disc catabolism and more full length aggrecan in aged
PAPPA
−/− mice compared to aged WT mice. Additionally, further studies should confirm whether young
PAPPA
−/− mice also have decreased GAG content or this loss occurs during the aging process.
SIGNIFICANCE
The results of this study suggest the net effect of IGF‐1 regarding both anabolic and catabolic effects must be examined to fully understand if IGF‐1 is beneficial for disc homeostasis during age‐associated IDD.
Support or Funding Information
R01‐AG044376‐01 & Ferguson Foundation Grant
Qualitative and quantitative results show decreased disc proteoglycan content, including aggrecan, in aged
PAPPA
−
/
− mice.
Figure 1
Despite less proteoglycan content, aged PAPPA −/− mice had less fragmented aggrecan and lower levels of catabolic markers, MMP‐3 and ADAMTS‐4 compared to aged WT mice.
Figure 2
Fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis elicit spontaneous proliferation of autologous T cells in an HLA-DR and CD47 costimulation-dependent manner. T cell ...costimulation through CD47 is attributed to specific interaction with thrombospondin-1 (TSP1), a CD47 ligand displayed on FLS. CD47 binding by FLS has broad biological impact that includes adhesion and the triggering of specific costimulatory signals. TSP1(+) FLS are highly adhesive to T cells and support their aggregation and growth in situ. Long-term cultures of T cells and FLS form heterotypic foci that are amenable to propagation without exogenous growth factors. T cell adhesion and aggregate formation on TSP1(+) FLS substrates are inhibited by CD47-binding peptides. In contrast, FLS from arthroscopy controls lack adhesive or T cell growth-promoting activities. CD47 stimulation transduces a costimulatory signal different from that of CD28, producing a gene expression profile that included induction of ferritin L chain, a component of the inflammatory response. Ferritin L chain augments CD3-induced proliferation of T cells. Collectively, these results demonstrate the active role of FLS in the recruitment, activation, and expansion of T cells in a CD47-dependent manner. Because TSP1 is abundantly expressed in the rheumatoid synovium, CD47-TSP1 interaction is proposed to be a key component of an FLS/T cell regulatory circuit that perpetuates the inflammatory process in the rheumatoid joint.
Changes in T cell populations and concomitant perturbation of T cell effector functions have been postulated to account for
many aging-related immune dysfunctions. Here, we report that high ...frequencies of CD28 null CD4+ T cells were found in elderly individuals. Because deviations in the function of these unusual CD4+ T cells might be
directly related to CD28 deficiency, we examined the molecular basis for the loss of CD28 expression in CD4+ T cells. In reporter
gene bioassays, the minimal promoter of the CD28 gene was mapped to the proximal 400 base pairs (bp) of the 5â² untranslated region. CD28 deficiency was associated with the
loss of two noncompeting binding activities within a 67-bp segment of the minimal promoter. These binding activities were
not competed by consensus Ets , Elk , or AP3 motifs that were found within the sequence stretch. The DNA-protein complexes were also not recognized by antibodies to Ets-related
transcription factors. Furthermore, introduction of mutations into the 67-bp segment at positions corresponding to the two
DNA-protein interaction sites, i.e. nucleotides spanning â206 to â179 and â171 to â148, resulted in the loss of specific nuclear factor binding activities and
the abrogation of promoter activity. These observations implicate at least two regulatory motifs in the constitutive expression
of CD28. The loss of binding activity of trans -acting factors specific for these sequences may contribute to the accumulation CD4+CD28 null T cells during aging.
Activation of circulating monocytes in patients with acute coronary syndromes may reflect exposure to bacterial products or stimulation by cytokines such as IFN-gamma. IFN-gamma induces ...phosphorylation and nuclear translocation of transcription factor STAT-1, which initiates a specific program of gene induction. To explore whether monocyte activation is IFN-gamma driven, patients with unstable (UA) or stable angina (SA) were compared for nuclear translocation of STAT-1 complexes and upregulation of IFN-gamma-inducible genes CD64 and IP-10.
Peripheral blood mononuclear cells were stained for expression of CD64 on CD14(+) monocytes and analyzed by PCR for transcription of IP-10. Expression of CD64 was significantly increased in patients with UA. Monocytes from UA patients remained responsive to IFN-gamma in vitro, with accelerated transcriptional competency of CD64. IP-10-specific sequences were spontaneously detectable in 82% of the UA patients and 15% of SA patients (P<0.001). Most importantly, STAT-1 complexes were found in nuclear extracts prepared from freshly isolated monocytes of patients with UA, which provides compelling evidence for IFN-gamma signaling in vivo.
Monocytes from UA patients exhibit a molecular fingerprint of recent IFN-gamma triggering, such as nuclear translocation of STAT-1 complexes and upregulation of IFN-gamma-inducible genes CD64 and IP-10, which suggests that monocytes are activated, at least in part, by IFN-gamma. IFN-gamma may derive from stimulated T lymphocytes, which implicates specific immune responses in the pathogenesis of acute coronary syndromes.
Thrombospondin-1 (TSP) is a transiently expressed matricellular protein known to promote chemotaxis of leukocytes to inflammatory sites. However, TSP and its receptor CD36 are abundantly expressed in ...chronically inflamed tissues such as the rheumatoid synovium. Here, we show that TSP provides the costimulatory signal that is necessary for the activation of autoreactive T cells. Data presented reveal that TSP-mediated costimulation is achieved through its independent interaction with CD36 on APCs and with CD47 on T cells. We propose that a CD47-TSP-CD36 trimolecular complex is a novel costimulatory pathway that significantly decreases the threshold of T cell activation. Consistent with the paradigm that lesions in rheumatoid synovitis are sites of antigenic recognition, the characteristic focal expression of TSP on APCs such as macrophages and fibroblast-like synoviocytes suggest a central role of TSP in the expansion of tissue-infiltrating T cells.
Systemic-onset juvenile idiopathic arthritis (SJIA) is a disease of unknown etiology with an unpredictable response to treatment. We examined two groups of patients to determine whether there are ...serum protein profiles reflective of active disease and predictive of response to therapy. The first group (n = 8) responded to conventional therapy. The second group (n = 15) responded to an experimental antibody to the IL-6 receptor (MRA). Paired sera from each patient were analyzed before and after treatment, using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). Despite the small number of patients, highly significant and consistent differences were observed before and after response to therapy in all patients. Of 282 spectral peaks identified, 23 had mean signal intensities significantly different (P < 0.001) before treatment and after response to treatment. The majority of these differences were observed regardless of whether patients responded to conventional therapy or to MRA. These peaks represent potential biomarkers of active disease. One such peak was identified as serum amyloid A, a known acute-phase reactant in SJIA, validating the SELDI-TOF MS platform as a useful technology in this context. Finally, profiles from serum samples obtained at the time of active disease were compared between the two patient groups. Nine peaks had mean signal intensities significantly different (P < 0.001) between active disease in patients who responded to conventional therapy and in patients who failed to respond, suggesting a possible profile predictive of response. Collectively, these data demonstrate the presence of serum proteomic profiles in SJIA that are reflective of active disease and suggest the feasibility of using the SELDI-TOF MS platform used as a tool for proteomic profiling and discovery of novel biomarkers in autoimmune diseases.
Replicative senescence of human T cells is characterized by the loss of CD28 expression, exemplified by the clonal expansion of CD28(null) T cells during repeated stimulation in vitro as well as in ...chronic inflammatory and infectious diseases and in the normal course of aging. Because CD28 is the major costimulatory receptor for the induction of T cell-mediated immunity, the mechanism(s) underlying CD28 loss is of paramount interest. Current models of replicative senescence involve protracted procedures to generate CD28(null) cells from CD28(+) precursors; hence, a T-cell line model was used to examine the dynamics of CD28 expression. Here, we show the versatility of the JT and Jtag cell lines in tracking CD28(null) <--> CD28(hi) phenotypic transitions. JT and Jtag cells were CD28(null) and CD28(lo), respectively, but expressed high levels of CD28 when exposed to phorbol 12-myristate 13-acetate. This was a result of the reconstitution of the CD28 gene transcriptional initiator (INR). Tumor necrosis factor-alpha reduced CD28 expression because of the inhibition of INR-driven transcription. Ligation of CD28 by an antibody or by CD80 also down-regulated CD28 transcription through the same mechanism, providing evidence that CD28 can generate a T cell receptor-independent signal with a unique biological outcome. Collectively, these data unequivocally demonstrate the critical role of the INR in the regulation of CD28 expression. T cell lines with transient expression of CD28 are invaluable in the dissection of the biochemical processes involved in the transactivation of the CD28 INR, the silencing of which is a key event in the ontogenesis of senescent T cells.
We recently reported that aging is accompanied by the emergence of CD4+CD28null T cells, a functionally aberrant lymphocyte subset rarely seen in individuals younger than 40 years. Here, we directly ...examined whether the lack of CD28 expression is due to a defect at the level of transcriptional initiation. Molecular studies reveal that CD28 gene transcription is controlled by two sequence motifs, sites α and β. In vitro transcription assays using initiator-dependent DNA templates revealed that reversed polarity or the deletion of either motif inhibited transcription, indicating that α/β sequences constitute a composite initiator. Moreover, nuclear extracts from CD28null cells failed to activate transcription of αβ-initiator DNA templates. Transcription of such templates was, however, restored with the addition of extracts from CD28+cells. Although previously described initiator elements have been defined by a consensus sequence, the αβ-initiator has no homology to such sequence. These studies demonstrate that initiators have functions other than positioning elements for the basal transcription complex. Rather, initiators can have a direct role in regulating the expression of specific genes. The gain or loss of initiator activity can be an important determinant of cell phenotypes.
Aging and chronic inflammatory syndromes, such as rheumatoid arthritis, are associated with high frequencies of CD4(+)CD28(null) T cells, which are rarely seen in healthy individuals younger than 40 ...years. Inasmuch as rheumatoid arthritis and aging are also associated with elevated levels of TNF-alpha, we examined whether this proinflammatory cytokine influences CD28 expression. Incubation of T cell lines and clones as well as Jurkat cells with TNF-alpha induced a reduction in the levels of cell surface expression of CD28. This effect of TNF-alpha was reversible; however, continuous culture of CD4(+)CD28(+) T cell clones in TNF-alpha resulted in the appearance of a CD28(null) subset. In reporter gene bioassays, TNF-alpha was found to inhibit the activity of the CD28 minimal promoter. Inactivation of the promoter was accompanied by a marked reduction in DNA-protein complex formation by two DNA sequence motifs corresponding to the transcriptional initiator of the CD28 gene. Indeed, in vitro transcription assays showed that nuclear extracts from TNF-alpha-treated cells failed to activate transcription of DNA templates under the control of a consensus TATA box and the CD28 initiator sequences. In contrast, similar extracts from unstimulated T cells supported transcription. These results demonstrate that TNF-alpha directly influences CD28 gene transcription. We propose that the emergence of CD4(+)CD28(null) T cells in vivo is facilitated by increased production of TNF-alpha.
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