Impaired T cell capping and receptor regeneration in active systemic lupus erythematosus. Evidence for a disorder intrinsic to the T lymphocyte.

It is currently unclear whether the T cell dysfunctions observed during active systemic lupus erythematosus (SLE) reflect a disorder intrinsic to the T cell or defects that result from interaction with anti-T cell autoantibody. To determine whether a disorder intrinsic to the T cell exists in SLE, the T cell capping mechanism was selected as a model of cellular function. The normal T cell capping mechanism is a rapid, energy-dependent and coordinated sequence of membrane events that consists of microaggregation, capping, endocytosis, and regeneration of the surface molecule. The monoclonal antibodies OKT3, OKT4, and OKT8, directed against the T cell-specific membrane glycoproteins T-3, T-4, T-8, served as specific probes of the glycoproteins' mobility within the membrane and membrane glycoprotein regeneration. When compared with greater than 91% T cell capping in normal and control subjects with active Sjögren's syndrome, active rheumatoid arthritis and active tuberculosis, only 49-60% of T cells from active SLE patients completed the capping sequence (SLE vs. healthy controls; T-3, P less than 0.002; T-4, P less than 0.004; T-8, P less than 0.002). Colchicine (10(-5) M), which inhibits microtuble polymerization and augments the rate of normal T cell capping, failed to restore the abnormal capping. However, as judged by the elapsed time intervals to half-maximal capping, the capping kinetics of the T cells able to initiate capping were not significantly different from controls. Fluorescence microscopy demonstrated an abnormal staining pattern characterized by microaggregation of ligand-glycoprotein complexes on resting T cells, coarse aggregation of ligand-glycoprotein complexes over the surfaces of cells that failed to cap, and cleaved or disrupted caps. After clearance of determinants by capping, greater than 94% of T cells from healthy controls regenerated T-3, -4, and -8 within 24 h. In contrast, only 20-40% of capped T cells from active SLE patients reexpressed new determinants. With improving disease activity, the proportion of cells capping and regenerating T-3, -4, and -8 increased, but remained significantly below control levels. In conclusion, this study has identified a disorder of T cell surface glycoprotein mobility and regeneration affecting the majority (60-80%) of both the T-3+, T-4+, (inducer/helper), and T-3+, T-8+ (suppressor) subsets during active SLE. Although the impaired capping and reexpression improve with disease remission, a residual defect persists. The data support the concept of a disorder intrinsic to the T cell in SLE.

Early events of human T lymphocyte activation are associated with type I protein kinase A activity.

Human T lymphocytes possess both the type I and II isozymes of protein kinase A (PKA). The type I (PKA-I) isozyme is predominantly associated with the plasma membrane, whereas the type II (PKA-II) isozyme is primarily localized to the cytosol. Because the functions of both PKA-I and PKA-II isozymes in the biochemical events of T lymphocyte activation have not been clearly elucidated, we tested the hypothesis that very early events of normal human T lymphocyte activation are mediated by the PKA-I and/or PKA-II isozyme(s). Fresh normal human T cells and a normal human CD4+ T cell line (GK606) activated with anti-CD3-epsilon and recombinant interleukin 1 alpha (rIL-1 alpha) exhibited a peak six- to sevenfold increase of PKA phosphotransferase activity at 5 min that returned to baseline by 60 min. Similarly, both fresh T cells and the T cell line activated by phorbol myristate acetate and ionomycin demonstrated a peak eightfold increase of PKA activity by 15 min that returned toward baseline by 60 min. Chromatographic separation of the PKA isozymes and quantification of phosphotransferase activities after T cell activation by either agonist pair showed preferential activation of the PKA-I isozyme, resulting in a significant reduction in the ratio of PKA-I to PKA-II isozyme activity from 3.1:1-6.2:1 to 1.1:1-3.2:1. PKA-I isozyme activation resulted in the release of free catalytic (C) subunit, an increase in C subunit phosphotransferase activity, and the phosphorylation of T cell plasma membrane-associated proteins, p14, p17, p20, p21, p38, and p48. However, activation of the PKA-I isozyme did not appear to be required for the transcription of IL-2 mRNA, an event necessary for mitosis. These data indicate that ligand-induced T cell activation is associated with rapid activation of the PKA-I, but not PKA-II, isozyme that results in phosphorylation of plasma membrane-associated proteins. The involvement of the PKA-I isozyme during the very early events of T cell activation suggests that this isozyme may be an antigen- or mitogen-stimulated protein kinase.

Deficient type I protein kinase A isozyme activity in systemic lupus erythematosus T lymphocytes.

Systemic lupus erythematosus (SLE) is an autoimmune disorder of indeterminate etiology characterized by a dysfunctional cellular immune response. We have previously identified a metabolic disorder of the adenylate cyclase/cAMP/protein kinase A (AC/cAMP/PKA) pathway characterized by impaired cAMP-inducible, PKA-catalyzed protein phosphorylation in intact T lymphocytes from subjects with severe SLE disease activity. Because this metabolic disorder may contribute to abnormal T cell immune effector functions, we tested the hypothesis that impaired PKA-dependent protein phosphorylation is the result of a PKA isozyme deficiency in SLE T lymphocytes. Compared with healthy and rheumatoid arthritis (RA) controls, subjects with severe SLE activity exhibited reduced PKA-catalyzed phosphorylation of proteins in the T lymphocyte plasma membrane where the type I isozyme of PKA (PKA-I) is predominantly localized. Both silver staining and biosynthetic labeling of membrane-associated proteins with [35S]methionine demonstrated that reduced protein phosphorylation was not due to either an altered distribution of or absence of proteins. Moreover, phosphorylation of SLE membrane-associated proteins with the PKA catalytic (C) subunit showed a similar distribution and extent of phosphorylation compared with membrane proteins from healthy T cells, suggesting that SLE T cell membrane proteins could be phosphorylated. Sequential column chromatography of the type I and type II isozymes of PKA (PKA-I, PKA-II) demonstrated a deficiency of PKA-I isozyme activity. Compared with a ratio of PKA-I to PKA-II activity of 4.2:1 in healthy T cells, the activity ratio in T cells from subjects with severe SLE disease activity was 0.99:1 (P = 0.01, SLE versus healthy controls for PKA-I). The deficient PKA-I activity was associated with a significant increase of free C-subunit activity (P = 0.04, SLE versus healthy controls for C-subunit). T cells from subjects with mild/moderate SLE disease activity also exhibited diminished PKA-I activity, yielding a ratio of PKA-I to PKA-II activity of 2.4:1. By contrast, T cells from RA controls possessed increased PKA-I, PKA-II, and free C-subunit activities compared with healthy controls, resulting in a ratio of PKA-I to PKA-II activity of 3.6:1. We conclude that the reduced PKA-catalyzed protein phosphorylation in the plasma membrane of SLE T cells is the result of deficient PKA-I isozyme activity. This is the first identification of a deficiency of PKA activity in SLE T lymphocytes; the deficiency, resulting in diminished protein phosphorylation, may alter cellular homeostasis, contributing to the cellular immune dysfunctions observed in SLE.

Secretion of an articular cartilage proteoglycan-degrading enzyme activity by murine T lymphocytes in vitro.

Destruction of articular cartilage is the hallmark of inflammatory arthritides. Enzymes elaborated by mononuclear cells infiltrating the synovium mediate, in part, the degradation of the cartilage extracellular matrix. Since mononuclear cells are the dominant cell type found in chronic inflammatory synovitis, we investigated whether interaction of immune mononuclear cells with antigen initiated the synthesis and secretion of a proteoglycan-degrading enzyme activity. Proteoglycan-degrading enzyme activity was monitored by the capacity of murine spleen cell conditioned medium to release [3H]serine/35SO4 incorporated into rabbit cartilage proteoglycan monomer fraction (A1D1), and by the relative change in specific viscosity of bovine nasal cartilage proteoglycan monomer. The results demonstrated that both virgin and immune mononuclear cells spontaneously generated proteoglycan-degrading enzyme activity and that cellular activation and proliferation induced by the antigen keyhole limpet hemocyanin or the mitogen phytohemagglutinin was not required. Kinetic studies demonstrated stable release of the enzyme activity over 72 h. Cell separation studies showed that T lymphocytes, a thymoma line, and macrophages separately produced proteoglycan-degrading enzyme activity. The enzyme activity has been partially characterized and appears to belong to a class of neutral pH metal-dependent proteinases. These observations, the first to demonstrate that T lymphocytes secrete an enzyme capable of degrading cartilage proteoglycan, raise the possibility that this enzyme activity contributes to cartilage extracellular matrix destruction in vivo. Moreover, these data support the conclusion that production of this enzyme by T lymphocytes is independent of an antigen-specific stimulus.

B cells from patients with systemic lupus erythematosus display abnormal antigen receptor-mediated early signal transduction events.

To understand the molecular mechanisms that are responsible for the B cell overactivity that is observed in patients with SLE, we have conducted experiments in which the surface immunoglobulin (sIg)-mediated early cell signaling events were studied. The anti-sIgM-mediated free intracytoplasmic calcium ([Ca2+]i) responses were significantly higher in SLE B cells compared with responses of normal individuals and to those of patients with other systemic autoimmune rheumatic diseases. The anti-IgD mAb induced [Ca2+]i responses were also higher in lupus B cells than in controls. The magnitude of anti-sIgM-mediated Ca2+ release from intracellular stores was also increased in B cells from SLE patients compared with normal controls. The amount of inositol phosphate metabolites produced upon crosslinking of sIgM was slightly higher in patients with lupus than in normal controls, although the difference was not statistically significant. In contrast, the degree of anti-sIgM-induced protein tyrosine phosphorylation was obviously increased in lupus patients. Our study demonstrates clearly for the first time that SLE B cells exhibit aberrant early signal transduction events, including augmented calcium responses after crosslinking of the B cell receptor and increased antigen-receptor-mediated phosphorylation of protein tyrosine residues. Because the above abnormalities did not correlate with disease activity or treatment status, we propose that they may have pathogenic significance.

UVB radiation and human monocyte accessory function: differential effects on pre-mitotic events in T-cell activation.

Purified T lymphocytes fail to proliferate in response to antigenic and mitogenic stimuli when cultured in the presence of accessory cells that have been exposed in vitro to sublethal doses of UVB radiation. Because proliferation represents a final stage in the T-cell activation process, the present study was conducted to determine whether T cells were able to progress through any of the pre-mitotic stages when UVB-irradiated monocytes were used as model accessory cells. In these experiments, monoclonal anti-CD3 antibodies were employed as the mitogenic stimulus. Culture of T cells with UVB-irradiated monocytes did allow the T cells to undergo an increase in intracellular free calcium, which is one of the first steps in the activation sequence. The T cells expressed interleukin-2 receptors, although at a reduced level. However, T cells failed to produce interleukin-2 above background levels when they were placed in culture with monocytes exposed to UVB doses as low as 50 J/m2. Incubation of T cells with UVB-irradiated monocytes did not affect the subsequent capacity of T cells to proliferate, since they developed a normal proliferative response in secondary culture when restimulated with anti-CD3 antibodies and unirradiated monocytes. These studies indicate that T lymphocytes become partially activated when cultured with UVB-irradiated monocytes and mitogenic anti-CD3 monoclonal antibodies. In addition, they suggest that interleukin-2 production is the T-cell activation step most sensitive to inhibition when UVB-irradiated monocytes are employed as accessory cells.

Intact proteoglycan is a polyclonal activator of murine B-lymphocytes.

Destruction of articular cartilage is the hallmark of both inflammatory and degenerative arthritides. Since degradation of cartilage results in the release of proteoglycan (PG) monomers and fragments into the synovial fluid, the present study was initiated to determine whether hyaline cartilage PG can induce a cellular immune response. Nonimmune spleen cells obtained from A/St and C57Bl/6 mice were cultured with a crude extract of bovine nasal cartilage, a PG aggregate fraction, PG monomer or degraded PG monomer for varying time periods. Only intact PG monomer induced a proliferative response which peaked at day 2. The responding cell was a B-lymphocyte which did not require T-helper cell activity. Our results suggest that intact PG monomer is a polyclonal activator of B-lymphocytes.

Systemic lupus erythematosus in the elderly.

Aging modifies the clinical presentation and course of autoimmune disorders, although the mechanisms by which this occurs remain to be determined. Current evidence cited above supports the general concept that there is a natural senescence of the immune system. This evidence would suggest that somehow senescence directly affects gene expression, resulting in biochemical abnormalities that culminate in T-cell immunodysfunctions. This may be a principal factor that attenuates the autoimmune response to self-antigen and, therefore, the disease course. The authors speculate that there is a disorder primary to the T cell in SLE that is expressed as abnormal immunologic responses to self-antigens, resulting in autoimmunity. Although understanding of this primary T cell disorder is still limited, clinicians now know that the T cell harbors abnormal signaling pathways that reflect defective biochemical functions and seem to be genetically regulated. This aberrant signaling would be anticipated to affect both principal T cell subsets. It may hinder the capacity of cells, such as CD8 T cells, to effectively down-regulate the response of autoreactive CD4 helper T cells to autoantigens. Loss of self-regulation would manifest itself as loss of tolerance, a fundamental component of autoimmunity. The future challenge is to understand how aberrant signaling leads to loss of tolerance. Given this underlying genetic susceptibility in an aged individual whose T cells also are undergoing natural senescence, the authors suggest that it is conceivable that a stress factor may tip the balance in the favor of clinical disease. One such factor may be unspecified environmental stimuli. Yet another consideration is an intercurrent illness, such as an infection. It remains to be determined, however, what these environmental stimuli are and how they impact on the immune system to trigger disease.

The effect of nonsteroidal anti-inflammatory drugs on cAMP-dependent protein kinase-mediated phosphorylation by human chondrocytes in culture.

Although a principal pharmacological action of nonsteroidal anti-inflammatory drugs (NSAIDs) is to blunt eicosanoid synthesis by inhibiting cyclooxygenase, recent evidence indicates that NSAIDs may also interact directly with proteins that control the activity of adenylyl cyclase. Since the only physiological mechanism governing the action of cyclic AMP occurs via activation of its receptor, cyclic AMP-dependent protein kinase (cAMPPk; Kinase A), we determined whether NSAIDs affect intracellular substrate phosphorylation dependent on cAMPPk. The incorporation of 32Pi into cellular proteins that are substrates for cAMPPk was determined in intact human non-arthritic, aged non-arthritic and osteoarthritic chondrocytes in the presence or absence of NSAIDs, namely, sodium meclofenamate, indomethacin, tiaprofenic acid and sodium salicyclate. The transfer of [32P]-ATP was employed to identify phosphoproteins in a membrane fraction prepared from chondrocyte homogenates in the presence or absence of these NSAIDs. The lowest concentration of NSAID was similar to NSAID concentrations achieved during therapy for the arthritides. In intact human chondrocyte strains, activation of cAMPPk by dibutyryl cAMP (dBcAMP) resulted in the phosphorylation of intracellular substrates with an apparent M(r) of 55kD, 42kD, 26kD, 25kD, 22kD, 21.5kD, 20.5kD, and 17kD when examined by autoradiography after 12.5% SDS/PAGE. The NSAIDs augmented or potentiated phosphorylation of these proteins which were cAMPPk-dependent. In the chondrocyte membrane fraction, protein phosphorylation by cAMP was mimicked by isobutylmethylxanthine or by the purified catalytic subunit of bovine cAMPPk. NSAIDs augmented chondrocyte phosphorylation in the chondrocyte membrane fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical expression of autoimmune diseases in older adults.

Aging modifies the clinical presentation and course of autoimmune disorders. In the older person who carries a complement of predisposing genes, environmental factors may alter a senescent immune system and trigger the onset of autoimmunity. In this article about autoimmune diseases of the older adult, the authors discuss the epidemiology, clinical presentations, laboratory and radiographic findings, and management of systemic lupus erythematosus, primary Sjogren's syndrome, and idiopathic inflammatory myopathies.

High prevalence of T cell type I protein kinase A deficiency in systemic lupus erythematosus.

OBJECTIVE: To estimate the prevalence of protein kinase A type I isozyme (PKA-I) deficiency in a cohort of systemic lupus erythematosus (SLE) patients, and to establish whether the isozyme deficiency is associated with SLE disease activity. METHODS: Thirty-five SLE patients and 35 age-, sex-, and race-matched normal controls were studied. Fifteen subjects were restudied on at least 3 occasions over a 4-year interval. Clinical disease activity was estimated by the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI), and the T cell activation markers CD25+ and HLA-DR+ were quantified by flow cytometry. PKA-I isozyme activities were quantified in enriched T cells. Statistical analyses were performed by Student's t-test, Mann Whitney U test, and Pearson product moment test. RESULTS: The mean PKA-I activity in SLE T cells (540 pmoles/minute/mg of protein) was significantly lower than that in control T cells (1,578 pmoles/ minute/mg of protein) (P<0.001). The prevalence of isozyme deficiency in this cohort was 80%. During a 4-year interval, PKA-I activities remained significantly reduced, whereas SLEDAI scores significantly improved. There was no relationship between deficient PKA-I activity and either SLEDAI scores or the proportion of T cells bearing CD25+ or HLA-DR+ activation markers. CONCLUSION: There is a high prevalence of deficient T cell PKA-I isozyme activity in SLE that persists over time and is independent of SLE disease activity.

Molecular aberrations in human systemic lupus erythematosus.

Systemic lupus erythematosus is an autoimmune disorder that predominantly affects women during the childbearing years. Clinically, major organ systems are affected, including the skin, kidneys and nervous system. Genetic, hormonal, environmental and immunoregulatory factors contribute to the highly variable expression of the disease. Impaired cellular and humoral immune responses reflect disordered biochemical and molecular functions that might be determined genetically. Enhanced understanding of these molecular abnormalities should enable development of new, effective therapeutic agents in the near future.

Activation of type I protein kinase A during receptor-mediated human T lymphocyte activation.

The experiments reported herein have characterized the signaling pathway leading to stimulation of type I protein kinase A isozyme (PKA-I) activity during the early events of Ag receptor-mediated T cell activation. Inhibitor studies demonstrated that receptor-initiated activation of nonreceptor protein tyrosine kinases, phosphorylation and activation of phospholipase C-gamma 1, and activation of protein kinase C occur temporally and precede PKA-I activation. Bypass of both the TCR/CD3 complex and IL-1R and direct activation of protein kinase C by a phorbol ester can also activate PKA-I. To confirm that PKA-I activation via the TCR/CD3 complex and IL-1R requires antecedent protein tyrosine kinase-catalyzed phosphorylation of phospholipase C-gamma 1, we used wild-type and CD45-deficient (mutant J45.01) Jurkat T cell lines. Unlike wild-type Jurkat T cells, the absence of CD45 tyrosine phosphatase resulted in the failure of receptor-mediated activation of PKA-I activity and of IL-2 mRNA transcription in the mutant J45.01 Jurkat cell line. In conclusion, our data support the concept that a signal derived from ligand binding to both the TCR/CD3 complex and IL-1R receptor mediates rapid activation of the PKA-I isozyme in primary T lymphocytes by sequential activation of an intracellular pathway comprised of CD45 phosphatase/protein tyrosine kinase/polyphosphoinositide/Ca2+/protein kinase C pathway rather than via the conventional surface receptor/stimulatory G protein system.

Impaired mobility of human T lymphocyte surface molecules during inactive systemic lupus erythematosus. Relationship to a defective cAMP pathway.

The T lymphocytes of patients with active systemic lupus erythematosus (SLE) exhibit impaired capping of the surface molecules CD3, CD4, and CD8 and a defective cAMP-dependent pathway. Since the mobility of these molecules is regulated in part by cAMP, we sought to determine whether there is a specific defect(s) along the T cell cAMP pathway that contributes to the persistent capping disorder observed during inactive SLE. The data suggest that a defect may exist at the level of cAMP-dependent protein kinase activation or at a point distally. We propose that a disorder of cAMP-dependent protein kinase activity might account for the defect of capping observed in both the CD3, CD4 (helper/inducer) and CD3, CD8 (suppressor) subsets observed in SLE.

Regulation of human T lymphocyte surface antigen mobility by purinergic receptors.

The present study was undertaken to establish whether the capping mechanism of normal human T lymphocytes is regulated by a purinergic receptor. Interaction of T lymphocytes with adenosine significantly increased the mobility of the T3, T4, and T8 surface antigens. This enhanced rate of capping was reflected by a significant decrement in the time intervals to achieve half-maximal capping. T lymphocytes preincubated with theophylline or isobutylmethylxanthine did not exhibit accelerated capping or a decrease in the time required for half-maximal capping in response to adenosine, suggesting that these agents inhibited the binding of adenosine to its receptor. A role for a cAMP-dependent pathway in capping was suggested by the observation that the phosphodiesterase inhibitor RO-201724 caused a decrease in the concentration of adenosine required to accelerate the capping process. Moreover, exposure of T lymphocytes to the cyclic nucleotide derivatives 8-N3-cAMP and 8-Br-cAMP mimicked the effect of adenosine, significantly reducing the time to half-maximal capping. Photoaffinity labeling of intracellular cAMP receptors with 32P-8-N3-cAMP indicated that adenosine caused occupancy of the receptors. This effect of adenosine was inhibited by theophylline, a known purinergic receptor blocker. The data support the concept that the T cell capping mechanism is mediated by an adenylate cyclase-coupled purinergic receptor that activates a cAMP-dependent pathway, and that this pathway is functional in the T3+, T4+, (inducer) and T3+, T8+ (suppressor) subsets.

mRNA mutations of type I protein kinase A regulatory subunit alpha in T lymphocytes of a subject with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disorder of indeterminate etiology characterized by multiple T lymphocyte immune effector dysfunctions. Protein kinase A (PKA) isozymes contribute to the regulation of T cell immune effector functions. In SLE T cells, there is a profound deficiency of PKA-I isozyme activity characterized by both reduced RI alpha transcript and RI alpha protein levels. To identify a molecular mechanism(s) for this isozyme deficiency, we utilized single-strand conformation polymorphism (SSCP) analysis to detect structural changes in the cDNA. Of 10 SLE subjects, cDNAs from a single subject revealed a shifted band. Sequence analyses demonstrated that a shifted SSCP band from SLE T cells carried heterogeneous transcript mutations, including deletions, transitions and transversions. Most of these transcript mutations are clustered adjacent to GAGAG motifs and CT repeats-regions that are susceptible to transcript editing and/or molecular misreading. By contrast, no genomic mutations were identified. These results suggest the occurrence of mRNA editing and/or defective function of RNA polymerase in a subject with SLE. Mutant RI alpha transcripts are pathophysiolgically significant, for they can encode diverse, aberrant RI alpha isoforms, including truncated, dominant-negative subunits, resulting in deficient PKA-I activity. We propose that deficient PKA-I isozyme activity contributes to the pathogenesis of SLE by hindering effective signal transduction and impairing T cell effector functions.