Racial and ethnic disparities in disease activity and function among persons with rheumatoid arthritis from university-affiliated clinics.

OBJECTIVE: Health outcomes in rheumatoid arthritis (RA) have improved significantly over the past 2 decades. However, research suggests that disparities exist by race/ethnicity and socioeconomic status, with certain vulnerable populations remaining understudied. Our objective was to assess disparities in disease activity and function by race/ethnicity and explore the impact of language and immigrant status at clinics serving diverse populations. METHODS: We examined a cross-sectional study of 498 adults with confirmed RA at 2 rheumatology clinics: a university hospital clinic and a public county hospital clinic. Outcomes included the Disease Activity Score in 28 joints (DAS28) and its components, and the Health Assessment Questionnaire (HAQ), a measure of function. We estimated multivariable linear regression models including interaction terms for race/ethnicity and clinic site. RESULTS: After adjusting for age, sex, education, disease duration, rheumatoid factor status, and medication use, clinically meaningful and statistically significant differences in DAS28 and HAQ scores were seen by race/ethnicity, language, and immigrant status. Lower disease activity and better function was observed among whites compared to nonwhites at the university hospital. This same pattern was observed for disease activity by language (English compared to non-English) and immigrant status (US-born compared to immigrant) at the university clinic. No significant differences in outcomes were found at the county clinic. CONCLUSION: The relationship between social determinants and RA disease activity varied significantly across clinic setting with pronounced variation at the university, but not at the county clinic. These disparities may be a result of events that preceded access to subspecialty care, poor adherence, or health care delivery system differences.

Socioeconomic determinants of disability and depression in patients with rheumatoid arthritis.

OBJECTIVE: To examine the relationship between functional limitation, socioeconomic inequality, and depression in a diverse cohort of patients with rheumatoid arthritis (RA). METHODS: The study design was cross-sectional and subjects were from the University of California, San Francisco RA Cohort. Patients were enrolled from 2 rheumatology clinics, an urban county public hospital and a university tertiary care medical center. Age, sex, race/ethnicity, disease activity, functional limitation, and medications were variables collected at clinical visits. The patient's clinic site was used as a proxy for his or her socioeconomic status. The outcome variable was depressive symptom severity measured by the Patient Health Questionnaire 9. Differences in characteristics between depressed and nondepressed patients were calculated using 2-sided t-tests or the Pearson's chi-square test. For the multivariate analysis, repeated measures with generalized estimating equations were used. RESULTS: There were statistically significant differences between depressed and nondepressed patients related to race/ethnicity, public versus tertiary care hospital rheumatology clinic, disability, and medications. In the multivariate analysis, increased functional limitation and public clinic site remained significantly associated with increased depression scores. A significant interaction existed between clinic site and disability. Mean depression scores rose more precipitously as functional limitation increased at the public hospital rheumatology clinic. CONCLUSION: There are disparities in both physical and mental health among individuals with low socioeconomic status. The psychological effects of disability vary in patients with RA such that a vulnerable population with functional limitations is at higher risk of developing depressive symptoms.

Predictors of depression in a multiethnic cohort of patients with rheumatoid arthritis.

OBJECTIVE: Patients with rheumatoid arthritis (RA) who experience depression have worse health outcomes. This study identifies predictors of depression in an ethnically and racially diverse population of patients with RA. METHODS: Patients with RA in a prospective cohort at the San Francisco General Hospital outpatient rheumatology clinic were included if they were age >or=18 years, met the American College of Rheumatology classification criteria for RA, had a Health Assessment Questionnaire (HAQ) score collected, and had the RA-specific Disease Activity Score performed by a rheumatologist. The outcome variable was a depression score measured by the Patient Health Questionnaire 9 (PHQ-9), a self-report questionnaire validated to correlate with a diagnosis of major depression. RESULTS: Three hundred forty-nine clinical visits for 172 patients were included in the analysis. Forty percent of patients scored >or=10 on the PHQ-9 during at least one clinic visit, which corresponds to a symptom severity of at least moderate depression. The mean PHQ-9 score was 7, corresponding to a symptom severity of mild depression. In the multivariate analysis, higher HAQ scores were associated with depression, and Asians had lower depression scores compared with Hispanic, white, and African American subjects. CONCLUSION: Identifying associated predictors of depression in a diverse population of patients with RA can help guide treatment, which should include preventing disability and decreased function as well as targeting depressive symptoms more specifically in patients with RA.

Analysis of inositol phospholipid turnover during lymphocyte activation.

Receptor-mediated activation of phospholipase C (PLC) leads to the hydrolysis of membrane inositol phospholipids, generating diacylglycerol (DAG) and water-soluble inositol phosphates. This signaling mechanism is used by antigen receptors on T and B cells that have been implicated as mediators of receptor-induced influx of extracellular Ca(2+). This unit provides protocols that describe the resolution of InsP by Dowex anion-exchange chromatography. This technique provides a reliable means of separating inositol monophosphate, inositol bisphosphate, and inositol trisphosphate, but does not resolve isomers of these. An Alternate Protocol describes the separation of inositol phosphates by anion-exchange HPLC. A protocol for resolution of inositol phospholipids by thin-layer chromatography (TLC) is also provided.

Cutting edge: tyrosine-independent transmission of inhibitory signals by CTLA-4.

CTLA-4 is an important inhibitor of T cell activation. We used Jurkat cells expressing mutants of murine CTLA-4 to study the structural requirements for inhibitory signaling. We find that signals for the inhibition of IL-2 secretion are delivered efficiently by a CTLA-4 mutant in which both cytoplasmic tyrosines have been replaced by phenylalanines. A CTLA-4 mutant that lacks the carboxyl-terminal half of the intracellular domain also retains the ability to inhibit, but deletion of an additional 11 aa completely abrogates that capability. We conclude that delivery of an inhibitory signal requires the membrane-proximal region of the CTLA-4 cytoplasmic domain and does not depend upon the tyrosine phosphorylation of CTLA-4.

Identification of tyrosine phosphorylation sites in the CD28 cytoplasmic domain and their role in the costimulation of Jurkat T cells.

The cytoplasmic domain of CD28 contains four tyrosine residues. Because signal transduction by CD28 appears to involve its tyrosine phosphorylation, we determined sites of CD28 tyrosine phosphorylation using mutants of mouse CD28 that retained tyrosine at one position, with the remaining three positions mutated to phenylalanine. When expressed in Jurkat cells and stimulated by mAb, only the mutants with tyrosine at position 170 or 188 were tyrosine phosphorylated. Phosphorylation of Tyr170 recruits phosphatidylinositol 3-kinase to CD28. Tyr188 has not been associated with any specific signaling event, but we found that ligation of CD28 by the natural ligand B7.2 also induced phosphorylation of Tyr188, suggesting that this event is of physiological importance. Consistent with that possibility, mutation of Tyr188 to phenylalanine severely impaired the ability of mouse CD28 to deliver a costimulus for the expression of CD69 and the production of IL-2. The functional consequences of the mutation of Tyr188 were unique; mutation of the other three tyrosines, individually or in combination, did not impair costimulation. Therefore, of the four CD28 tyrosine residues only Tyr188 is required for signaling in Jurkat cells, suggesting that its phosphorylation is a key event in the costimulation of T cells.

Mutational analysis of CD28-mediated costimulation of Jun-N-terminal kinase and IL-2 production.

The accessory molecule CD28 delivers a costimulus that acts in concert with TCR signals to promote T cell activation. Activation of Jun-N-terminal kinases (JNK) requires simultaneous stimulation of the TCR and CD28 and, therefore, likely plays an important role in signal integration during costimulation. We investigated the effects of mutations in the 41-amino acid cytoplasmic domain of murine CD28 on its ability to deliver costimuli for JNK activation and IL-2 production when expressed in Jurkat T cells. Our results indicate that the costimulus for JNK activation requires the membrane-proximal 24 amino acids of the CD28 cytoplasmic domain and is not mediated by the tyrosine-based recruitment of signaling molecules, including phosphatidylinositol 3-kinase. Deletion of the carboxyl-terminal 17 amino acids does not affect the ability of CD28 to augment JNK activation but impairs its ability to enhance TCR-mediated production of IL-2, demonstrating that optimal costimulation of IL-2 production requires CD28 signals in addition to the activation of JNK.

Phosphatidylinositol 3-kinase is required for CD28 but not CD3 regulation of the TEC family tyrosine kinase EMT/ITK/TSK: functional and physical interaction of EMT with phosphatidylinositol 3-kinase.

Ligation of the TCR or CD28 induces activation of phosphatidylinositol 3-kinase (PI3K), the TEC family protein tyrosine kinase, EMT/ITK/TSK (EMT), and the SRC family tyrosine kinase, LCK. LCK is required for the activation and phosphorylation of EMT induced by ligation of the TCR or CD28 placing LCK upstream of EMT in T cell signaling cascades. We report herein that inhibition of PI3K activity with the specific inhibitors LY294002 and wortmannin markedly decreased EMT activation induced by CD28 cross-linking but not by CD3 cross-linking. Further, inhibition of PI3K markedly decreased EMT in vitro autokinase activity induced by activated LCK. In contrast, PI3K inhibitors did not alter CD28 or CD3 cross-linking or LCK-induced EMT phosphorylation. Consistent with the requirement of PI3K activity for CD28 but not CD3-induced stimulation of the EMT in vitro autokinase activity, a small but significant portion of cellular EMT associates with PI3K following CD28 cross-linking but not following CD3 cross-linking. CD28-induced association of EMT with PI3K also requires functional expression of LCK. Fusion proteins containing the SRC homology 2 domain of EMT interact with PI3K or a PI3K-associated molecule in a tyrosine phosphorylation-dependent manner. Taken together, the data suggest that EMT is differentially regulated and recruited to different signaling complexes following ligation of CD28 or the TCR complex, perhaps contributing to the disparate roles that EMT appears to play downstream of CD28 and the TCR.

Efficient CD28 signalling leads to increases in the kinase activities of the TEC family tyrosine kinase EMT/ITK/TSK and the SRC family tyrosine kinase LCK.

Optimal T cell activation requires crosslinking of the T cell receptor (TCR) concurrently with an accessory receptor, most efficiently CD28. Crosslinking of CD28 leads to increased interleukin 2 (IL2) production, inhibition of anergy and prevention of programmed cell death. Crosslinking of CD28 leads to rapid increases in tyrosine phosphorylation of specific intracellular substrates including CD28 itself. Since CD28 does not encode an intrinsic tyrosine kinase domain, CD28 must activate an intracellular tyrosine kinase(s). Indeed, crosslinking of CD28 increases the activity of the intracellular tyrosine kinases EMT/ITK and LCK. The phosphatidylinositol 3-kinase (PI3K) and GRB2 binding site in CD28 is dispensable for optimal IL2 production in Jurkat T cells. We demonstrate herein that murine Y170 (equivalent to human Y173) in CD28 is also dispensable for activation of the SRC family tyrosine kinase LCK and the TEC family tyrosine kinase EMT/ITK. In contrast, the distal three tyrosines in CD28 are required for optimal IL2 production as well as for optimal activation of the LCK and EMT/ITK tyrosine kinases. The distal three tyrosines of CD28, however, are not required for recruitment of PI3K to CD28. Furthermore, PI3K is recruited to CD28 in JCaM1 cells which lack LCK and in which EMT/ITK is not activated by ligation of CD28. Thus optimal activation of LCK or EMT/ITK is not obligatory for recruitment of PI3K to CD28 and thus is also not required for tyrosine phosphorylation of the YMNM motif in CD28. Taken together the data indicate that the distal three tyrosines in CD28 are integral to the activation of LCK and EMT/ITK and for subsequent IL2 production.

Molecular cloning of the cDNA encoding pp36, a tyrosine-phosphorylated adaptor protein selectively expressed by T cells and natural killer cells.

Activation of T and natural killer (NK) cells leads to the tyrosine phosphorylation of pp36 and to its association with several signaling molecules, including phospholipase Cgamma-1 and Grb2. Microsequencing of peptides derived from purified rat pp36 protein led to the cloning, in rat and man, of cDNA encoding a T- and NK cell-specific protein with several putative Src homology 2 domain-binding motifs. A rabbit antiserum directed against a peptide sequence from the cloned rat molecule recognized tyrosine phosphorylated pp36 from pervanadate-treated rat thymocytes. When expressed in 293T human fibroblast cells and tyrosine-phosphorylated, pp36 associated with phospholipase Cgamma-1 and Grb2. Studies with GST-Grb2 fusion proteins demonstrated that the association was specific for the Src homology 2 domain of Grb-2. Molecular cloning of the gene encoding pp36 should facilitate studies examining the role of this adaptor protein in proximal signaling events during T and NK cell activation.

The CD28-B7 costimulatory pathway and its role in autoimmune disease.

The activation of naive CD4+ T cells requires two discrete signals: a signal delivered by the T cell receptor following recognition of antigen and an accessory signal transduced when costimulatory receptors interact with their ligands. Particularly important in the development of an immune response to foreign antigens is the T cell molecule CD28, which delivers a potent costimulus when engaged by ligands, B7-1 and B7-2, on antigen-presenting cells. It is interesting that blockade of B7 molecules, which disrupts interactions with CD28 and prevents delivery of the CD28 costimulus, also alters the immune responses to self antigens and prevents the development of clinical disease in murine models of systemic and organ-specific autoimmunity. Herein we review the roles of CD28 and its B7 ligands in the pathogenesis of autoimmunity, discuss efforts to treat animal models of autoimmunity by modifying the CD28 signal, and consider the mechanisms by which manipulation of the CD28 signal alters the course of experimental autoimmune disease.

CD28 mediates transcriptional upregulation of the interleukin-2 (IL-2) promoter through a composite element containing the CD28RE and NF-IL-2B AP-1 sites.

Mutagenesis studies have demonstrated the requirement for the CD28-responsive element (CD28RE) within the interleukin-2 (IL-2) promoter for transcriptional upregulation by CD28. Here, we demonstrate that CD28 responsiveness is conferred by a composite element containing both the CD28RE and the NF-IL-2B AP-1 sites (RE/AP). Mutations at either site within the RE/AP composite element abolish activity. The RE/AP composite element is a site for signal integration within the IL-2 promoter, since its activation is dependent on at least two separate signalling pathways being activated, through the T-cell receptor, CD28, and/or phorbol myristate acetate. Activation is maximal when all three signals occur simultaneously. By using a panel of CD28 cytoplasmic domain mutants, it was found that the transcriptional activation of the RE/AP composite element correlates exactly with the pattern of IL-2 secretion induced by these mutants upon stimulation. Similar to the upregulation of IL-2 secretion, the transcriptional upregulation of the RE/AP composite element by CD28 is FK506 insensitive. The pattern of activation of the RE/AP composite element is different from that observed for either an NFAT or consensus AP-1 site, implying that RE/AP represents a unique element. Using gel shift analysis, we demonstrate that stimulation by CD28 induces the association of the NF-kappaB family member c-Rel to the CD28RE within the RE/AP composite element. The transcriptional upregulation of IL-2 by CD28 appears, therefore, to be mediated through the RE/AP composite element, involving the association of c-Rel with the CD28RE.

Wortmannin, a phosphatidylinositol 3-kinase inhibitor, blocks antigen-mediated, but not CD3 monoclonal antibody-induced, activation of murine CD4+ T cells.

Perturbation of several distinct T cell molecules, including the CD3/TCR complex, CD7, and CD28, activates phosphatidylinositol 3-kinase (PI3-K), but a clear consensus on the role of PI3-K in T cell activation has yet to emerge. We report here that CD3 mAb-induced IL-2 production by CD4+ T cells from DO11.10 TCR-alphabeta-transgenic mice is refractory to the potent PI3-K inhibitor, wortmannin, demonstrating that activation under these conditions is independent of PI3-K. In marked contrast, wortmannin substantially inhibits IL-2 production elicited by Ag (OVA(323-339) peptide) presented by appropriate APCs (syngeneic B7+ B cell blasts) and blocks Ag-induced differentiation of naive CD4+ DO11.10 T cells into IL-4-producing cells. Wortmannin inhibits Ag-induced conjugate formation between T cells and B7+ B cell blasts. Because T cell activation by Ag requires stable interactions with APCs, this inhibitory effect on conjugate formation may underlie the ability of wortmannin to block Ag-induced IL-2 production and differentiation.

Turning off natural killers. Innate immunity.

Inhibitory receptors specific for class I molecules of the major histocompatibility complex regulate the cytolytic activity of natural killer cells. Recent studies suggest that recruitment of the protein tyrosine phosphatase SHP-1 enables these receptors to block natural killing.

Structural requirements for CD28-mediated costimulation of IL-2 production in Jurkat T cells.

Although under certain conditions an association with phosphatidylinositol 3'-kinase (PI3-K) appears to be critical for CD28 signaling, mutation of the PI3-K binding site (Tyr 170) does not alter the costimulatory ability of murine CD28 (mCD28) in Jurkat T cells. To define the structural requirements for this PI3-K-independent signaling, we expressed a series of mCD28 mutants in Jurkat. Mutation to Phe of all four cytoplasmic Tyr residues together (ALL F mutant) greatly reduced the ability of mCD28 to augment IL-2 production. Isolated re-constitution of Tyr 188, but not 170, 185, or 197, restored the ability of ALL F mCD28 to deliver a costimulus. Thus, a signal based upon Tyr 188 can deliver a costimulus for the enhancement of IL-2 production by Jurkat cells.

The SH3 domain of p56lck binds to proline-rich sequences in the cytoplasmic domain of CD2.

CD2, a cell surface glycoprotein expressed on T cells and natural killer cells, can couple to signaling pathways that result in T cell proliferation. An Src-like protein tyrosine kinase, p56lck, coprecipitates with CD2, and perturbation of CD2 by monoclonal antibodies results in an increase in the activity of p56lck, suggesting that an interaction with p56lck contributes to CD2-mediated signaling. Herein, we investigate the mechanism by which CD2 associates with p56lck. We demonstrate that CD2 and p56lck associate when coexpressed in nonlymphoid cells, that this association requires the cytoplasmic domain of CD2, and that the SH3 domain of p56lck mediates its interactions with CD2. Using truncation mutants of CD2, we identify two regions in the cytoplasmic domain of CD2 involved in binding p56lck. Each region contains a proline-rich sequence that, in the form of a synthetic peptide, directly binds p56lck. Thus, proline-rich sequences in the cytoplasmic domain of CD2 allow this transmembrane receptor to bind to the SH3 domain of p56lck.

CD28 delivers costimulatory signals independently of its association with phosphatidylinositol 3-kinase.

CD28, a cell-surface molecule expressed by T cells, delivers costimulatory signals during the activation of T cells by Ag. Stimulation of CD28 induces its association with phosphatidylinositol 3'-kinase (PI3-K), raising the possibility that PI3-K plays a critical role in CD28 signaling. We find, however, that wortmannin, a potent inhibitor of PI3-K, does not block CD28-mediated costimulation of Jurkat (a human T cell line) or of murine CD4+ T cells. To address further the role of PI3-K in CD28-mediated signaling, we expressed mutant murine CD28 molecules in Jurkat cells. Mutation of Tyr 170 of murine CD28 to Phe abrogates the association of murine CD28 with PI3-K but does not affect the ability of murine CD28 to augment IL-2 production by Jurkat cells in response to the combination of ionomycin and PMA. Conversely, a mutant of murine CD28 that has a Tyr at position 170 but has Phe substitutions at the remaining three cytoplasmic tyrosines retains the ability to associate with PI3-K and has an impaired ability to deliver a costimulus that augments IL-2 production. CD28, therefore, can deliver costimulatory signals independently of its interaction with PI3-K, and association with PI3-K is insufficient to mediate the full effector function of CD28. Optimal signaling by CD28 requires the integrity of one or more of the carboxyl-terminal three Tyr residues.

Intracellular signalling. Switching off signals.

In hematopoietic cells, the protein tyrosine phosphatase PTP1C appears to play a central role in the termination of signalling by receptors that activate protein tyrosine kinases.