Interactions of lymphocytes from patients with psoriatic arthritis or healthy controls and cultured endothelial cells

Psoriatic arthritis (PA) is an inflammatory rheumatic disease that can concomitantly occur in patients with psoriasis vulgaris. Psoriatic synovitis shows alterations of the synovial microvasculature. Inflammatory cells adhere to endothelial cells (EC) and migrate through the vascular wall of postcapillary venules located in the subintimal layer of the synovial membrane. The aim of our study was to investigate, first, the phenotype of lymphocytes (LC) of PA patients using flow cytometry (FC) with regard to activation antigens and adhesion molecules; second, the adhesion of LC of PA patients on cultivated resting or activated (with thrombin, LPS, IFN-gamma, or TNF-alpha) human umbilical vein endothelial cells (HUVEC) by counting the Feulgen-stained nuclei of both adherent LC and HUVEC using image analysis; and third, the synthesis of IL-6 and IL-8 in both LC and HUVEC 24 hr after cell contact. These cytokines were determined qualitatively by immunofluorescence and quantitatively at the single-cell level by FC as well as in the supernatants of the cultures using commercial cytokine ELISAs. Fourth, we investigated whether or not the LC adhesion on HUVEC as well as the cytokine production could be inhibited by monoclonal antibodies against LC- or EC-specific adhesion molecules. In contrast to controls PA patients showed an increased surface expression of CD11a, b, and c as well as of CD44 but a reduced surface expression of CD49d/CD29, and CD49e/CD29, and cell-bound fibronectin on CD3+ LC. The activation markers CD25 and HLA-DR were found to be slightly enhanced in PA. The cell adhesion was generally enhanced in PA patients vs controls. It could be reduced with monoclonal antibodies (MoAbs) against CD11a and CD18 on IFN-gamma- or TNF-alpha-activated HUVEC but was generally enhanced after treatment of HUVEC with MoAbs against CD54, CD62E, or CD106. Due to LC adhesion on HUVEC IL-6 and IL-8 were produced in significantly higher amounts in PA patients compared to controls. This effect occurred already in resting but was enhanced in activated HUVEC. While IL-6 is mainly produced by HUVEC but also in smaller quantities by LC, IL-8 is synthesized only by HUVEC and could be modified by preincubation with MoAbs against LC- or EC-specific adhesion molecules in parallel to the cell adhesion. The experiments show that the main adhesion pathway in LC homing of PA patients is the interaction of the LC adhesion molecule CD11a/CD18 with CD54 on EC followed by an enhanced synthesis of proinflammatory and chemotactic cytokines. These results favor the hypothesis that the pathological alterations of the microvasculature in PA patients are generated by altered homing processes.     

Increased incidence of apoptosis in transforming growth factor alpha-deficient mouse blastocysts

Platelets, activated by various agonists, produce microparticles (MP) from the plasma membrane, which are released into the extracellular space. Although the mechanism of MP formation has been clarified, their biological importance remains ill defined. We have recently shown that platelet-derived MP influence platelet and endothelial cell function. In this study, we have further examined the mechanism of cellular activation by platelet MP. To address the possibility that they may influence monocyte-endothelial interactions, we used an in vitro assay to examine their effects on the adhesion of monocytes to human umbilical vein endothelial cells (HUVEC). Platelet MP increased the adhesion of monocytes to HUVEC in a time- and dose-dependent manner. Maximal adhesion of monocytes to resting HUVEC was observed after 24 h of stimulation with MP. Similar kinetics were observed with U-937 (human promonocytic leukemia) cells, used as a model for the blood-borne monocyte. Maximal adhesion of resting monocytes to MP-stimulated HUVEC was observed after 5 h of stimulation with MP. The EC50s for MP-induced increases in HUVEC, monocyte, and U-937 cell adhesion is 8.74, 43.41, and 10.83 microg/ml of MP protein, respectively. The induction of monocyte-endothelial adhesion was mimicked by arachidonic acid isolated from MP. The observed increased cellular adhesiveness correlated with MP-induced upregulation of cell adhesion molecules. MP-stimulated HUVEC increased intracellular cell adhesion molecule-1 (ICAM-1) but not vascular cell adhesion molecule-1 (VCAM-1), P-, or E-selectin expression. Monocyte and U-937 lymphocyte function-associated antigen-1 (CD11a/CD18) and macrophage antigen-1 (CD11b/ CD18, alpham/beta2) were both upregulated upon MP stimulation, but an increase in p150,95 (CD11c/CD18), very late antigen-1, or ICAM-1 expression was not observed. The functional importance of these changes was demonstrated with blocking antibodies. MP also induced the chemotaxis of U-937 cells in a dose-dependent manner with an EC50 of 4.40 microg/ml of MP protein. Similarly, arachidonic acid isolated from MP mimicked the chemotactic response. A role for PKC was implicated in both adhesion and chemotaxis. GF 109203X, a specific inhibitor of PKC, significantly reduced monocyte-endothelial adhesion, as well as U-937 chemotaxis. The demonstration that platelet MP may modulate important aspects of endothelial and monocyte function provides a novel mechanism by which platelets may interact with such cells in human atherosclerosis and inflammation.     

Neutrophil adhesion molecule expression during cardiopulmonary bypass with bubble and membrane oxygenators

The neutrophil-mediated tissue injury associated with cardiopulmonary bypass (CPB) is thought to require the interaction of specific neutrophil and endothelial adhesion molecules. In this study, the effects of CPB on the expression of neutrophil CD11b and CD18 (the components of the Mac-1 adhesion molecule) were examined; the effects of membrane versus bubble oxygenators on the expression of neutrophil CD11b and CD18 were compared; and the plasma levels of the intercellular adhesion molecule-1 (cICAM-1), an inducible endothelial adhesion molecule, were measured. In addition, the time courses of complement activation and neutrophil granule release were measured to determine their temporal relationship to the expression of the neutrophil adhesion molecule. Fifteen adult patients underwent procedures requiring cardiopulmonary bypass; hollow-fiber membrane oxygenators were used in 8 (group M) and bubble oxygenators were used in 7 (group B). Blood samples were drawn before, during, and after CPB for determination of the expression of neutrophil CD11b and CD18 (immunofluorescent flow cytometry), and the plasma cICAM-1, elastase, lactoferrin (enzyme-linked immunoabsorbent assay), and plasma C3a (radioimmunoassay) levels. CPB caused an immediate and sustained increase in the neutrophil CD11b and CD18 expression in both groups; after 60 minutes of CPB, CD11b expression had increased by 116.9% +/- 19.1% in group B and by 79.3% +/- 8.5% in group M (p = 0.78). Over the same period, CD18 expression increased by 97.2% +/- 17.9% in group B and by 72.4% +/- 16.8% in group M (p = 0.67).(ABSTRACT TRUNCATED AT 250 WORDS)     

Porphyromonas gingivalis fimbriae use beta2 integrin (CD11/CD18) on mouse peritoneal macrophages as a cellular receptor, and the CD18 beta chain plays a functional role in fimbrial signaling

In this study, we demonstrate that Porphyromonas gingivalis fimbriae use molecules of beta2 integrin (CD11/CD18) on mouse peritoneal macrophages as cellular receptors and also show that the beta chain (CD18) may play a functional role in signalling for the fimbria-induced expression of interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) genes in the cells. Using a binding assay with 125I-labeled fimbriae, we observed that fimbrial binding to the macrophages was inhibited by treatment with CD11a, CD11b, CD11c, or CD18 antibody but not by that with CD29 antibody. Western blot assays showed that the fimbriae bound to molecules of beta2 integrin (CD11/CD18) on the macrophages. Furthermore, Northern blot analyses showed that the fimbria-induced expression of IL-1beta and TNF-alpha genes in the cells was inhibited strongly by CD18 antibody treatment and slightly by CD11a, CD11b, or CD11c antibody treatment. Interestingly, intracellular adhesion molecule 1 (ICAM-1), a ligand of CD11/CD18, inhibited fimbrial binding to the cells in a dose-dependent manner. In addition, ICAM-1 clearly inhibited the fimbria-induced expression of IL-1beta and TNF-alpha genes in the cells. However, such inhibitory action was not observed with laminin treatment. These results suggest the importance of beta2 integrin (CD11/CD18) as a cellular receptor of P. gingivalis fimbriae in the initiation stage of the pathogenic mechanism of the organism in periodontal disease.     

Endothelial and epithelial cells do not respond to complexes of peptidoglycan with soluble CD14 but are activated indirectly by peptidoglycan-induced tumor necrosis factor-alpha and interleukin-1 from monocytes

Peptidoglycan (PGN) activates macrophages through membrane CD14 (an endotoxin receptor) and binds to both soluble and membrane CD14. Since soluble CD14-lipopolysaccharide (LPS) complexes activate CD14-negative endothelial and epithelial cells, this study tested whether soluble CD14-PGN complexes activate human umbilical vein endothelial cells and epithelial-like U373 cells to secrete interleukin (IL)-6, express vascular cellular adhesion molecule-1, and translocate nuclear factor-kappaB. In contrast to LPS, endothelial, epithelial, and other cells of non-hemopoietic origin were unresponsive to PGN through soluble or membrane-bound CD14, whereas cells of hemopoietic origin were responsive to both PGN and LPS. PGN, similarly to LPS, activated endothelial and epithelial cells indirectly in the presence of 2%-4% blood, by inducing secretion of both tumor necrosis factor-alpha and IL-1 from monocytes. These results reveal different mechanisms of CD14 function and cell activation for LPS and PGN and also demonstrate strong indirect activation of endothelial and epithelial cells by both PGN and LPS.     

Interleukin 15 is produced by endothelial cells and increases the transendothelial migration of T cells In vitro and in the SCID mouse-human rheumatoid arthritis model In vivo

The capacity of endothelial cells (EC) to produce IL-15 and the capacity of IL-15 to influence transendothelial migration of T cells was examined. Human umbilical vein endothelial cells expressed both IL-15 mRNA and protein. Moreover, endothelial-derived IL-15 enhanced transendothelial migration of T cells as evidenced by the inhibition of this process by blocking monoclonal antibodies to IL-15. IL-15 enhanced transendothelial migration of T cells by activating the binding capacity of the integrin adhesion molecule LFA-1 (CD11a/CD18) and also increased T cell motility. In addition, IL-15 induced expression of the early activation molecule CD69. The importance of IL-15 in regulating migration of T cells in vivo was documented by its capacity to enhance accumulation of adoptively transferred human T cells in rheumatoid arthritis synovial tissue engrafted into immune deficient SCID mice. These results demonstrate that EC produce IL-15 and imply that endothelial IL-15 plays a critical role in stimulation of T cells to extravasate into inflammatory tissue.     

Local anesthetic lidocaine inhibits the effect of granulocyte colony-stimulating factor on human neutrophil functions

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) enhanced superoxide (O2-) release in human neutrophils stimulated by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) and inversely regulated the surface expression of cellular adhesion molecules, leukocyte adhesion molecule-1 (LAM-1) and CD11b/CD18 leukocyte integrin, on human neutrophils; that is, rhG-CSF downregulated the expression of LAM-1 and upregulated the expression of CD11b on neutrophils. The cationic local anesthetic lidocaine inhibited not only FMLP-induced O2- release in neutrophils but also FMLP-induced CD11b upregulation and LAM-1 downregulation on neutrophils in a dose-dependent manner. Lidocaine also abolished the priming effect of rhG-CSF for enhanced release of O2- in neutrophils and inhibited rhG-CSF-induced CD11b upregulation and LAM-1 downregulation on neutrophils in a dose-dependent manner. These findings suggest that lidocaine inhibits human neutrophil functions, such as adherence to endothelial cells, by interfering with the expression of cellular adhesion molecules on neutrophils, and that lidocaine might have anti-inflammatory properties by inhibiting the effect of inflammatory cytokines.     

Recent developments in peroxisome biology

Membrane molecules such as CD36 (OKM5), intercellular adhesion molecule-1 (ICAM-1, CD54), gamma interferon-induced protein 10 (gamma-IP10) and IL-1 are induced and/or upregulated in psoriatic epidermis. These molecules have important accessory, trafficking or signalling functions in the immune system and also play a role in the pathophysiology of psoriasis. The relevance of adhesion molecules, CD36 and epidermal IL-1 in psoriasis was studied in vitro in the autologous mixed epidermal cell - T lymphocyte reaction (MECLR). Their level of expression was quantitated in epidermal cell suspensions (ECS) from patients with psoriasis and their function was assessed by blocking with specific mAbs and antisera or by depleting CD36+ cells from the ECS prior to the MECLR. ECS from psoriatic lesions contained increased numbers of CD36+ (23 +/- 12%), ICAM-1(+) (31 +/- 14%) and IL-1(+) (57 +/- 21%) cells. The autologous MECLR was inhibited in samples from all patients by mAb to CD2 (LFA-2), CD11a (LFA-1alpha), CD18 (LFA-1beta), ICAM-1, CD58 (LFA-3) and an antiserum to IL-1beta. Thus, adhesion molecules facilitate inflammation in psoriasis not only via adhesion and recruitment of T lymphocyte in psoriatic lesions, but also via activation of T cells. Furthermore CD36 molecules on psoriatic epidermal cells do not costimulate autologous T lymphocytes in psoriasis. The observed costimulatory function of IL-1beta in the MECLR emphasizes its relevance in psoriasis.     

Are circulating neutrophils intravascularly activated in patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides?

Vascular injury in vasculitis may be due to activation of circulating neutrophils resulting in their increased adhesiveness to locally activated endothelium (Shwartzman phenomenon). Previously, we demonstrated up-regulation of endothelial intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in biopsies from patients with ANCA-associated vasculitis. In the present study, we investigated the expression of adhesion molecules (CD11b, ICAM-1, VLA-4, L-selectin) and activation markers (CD66b, CD64, CD63) on circulating neutrophils from patients with ANCA-associated vasculitis in comparison with their expression on cells from healthy volunteers and patients with sepsis. We related these findings to parameters of disease activity. Surface marker expression was determined by using a non-activating whole blood flow cytometric assay. The expression of activation markers, but not the expression of adhesion molecules, was increased on neutrophils from patients with active vasculitis. The expression of CD63 and CD66b on neutrophils correlated with disease activity as determined by the Birmingham Vasculitis Activity Score (BVAS). In contrast to patients with active vasculitis, patients with sepsis showed up-regulation of all markers, including adhesion molecules, suggesting that circulating neutrophils are fully activated in sepsis. We conclude that in ANCA-associated vasculitis, circulating neutrophils are not fully activated, since they do not express increased levels of adhesion molecules as sepsis or in the Shwartzman reaction. These findings are compatible with the concept that in vivo vascular damage in ANCA-associated vasculitides does not occur due to a Shwarzman-like reaction but only after ANCA-induced neutrophil activation at the endothelial cell surface.     

The Fcgamma receptor-mediated respiratory burst of rolling neutrophils to cytokine-activated, immune complex-bearing endothelial cells depends on L-selectin but not on E-selectin

Intracellular H2O2 generation, as a measure of the respiratory burst, was determined after stimulation of neutrophils by immune complex (IC)-bearing human umbilical vein endothelial cells. Under static conditions, neutrophils basically responded to the immune deposits on resting endothelial cells. The rotating shear forces of approximately 0.7 dynes/cm2, corresponding to the physiological flow in postcapillary venules, completely abolished this basal H2O2 generation. After activation of the IC-bearing endothelial layers with interleukin-1 (IL-1) or tumor necrosis factor (TNF), or both, for 4 hours, rolling adhesion of the neutrophils was induced, accompanied by considerable H2O2 production. The neutrophil respiratory burst was prominently inhibited by anti-FcgammaRIII MoAb 3G8 (72.4%), and partially by MoAb 2E1 against FcgammaRII (38.5%). Both MoAbs together inhibited the Fc-mediated H2O2 generation by 93. 4%. The respiratory burst and rolling adhesion were markedly blocked by MoAb LAM1-3 against L-selectin (91.3%), whereas the nonfunctional anti-L-selectin MoAb LAM1-14 was ineffective. F(ab)2' fragments of MoAb 7A9 against E-selectin inhibited neutrophil rolling by 98.6%, but not the respiratory burst. Moreover, rolling adhesion of neutrophils and the related oxidative burst were CD11b/CD18- independent. In summary, L-selectin has a unique auxiliary function in triggering the FcgammaR-mediated respiratory burst of rolling neutrophils to IC-bearing endothelial cells, thereby substituting CD11b/CD18 under conditions of flow.     

Cell adhesion molecules--update

Cell adhesion molecules are glycoproteins expressed on the cell surface and play an important role in inflammatory as well as neoplastic diseases. There are four main groups: the integrin family, the immunoglobulin superfamily, selectins, and cadherins. The integrin family has eight subfamilies, designated as beta 1 through beta 8. The most widely studied subfamilies are beta 1 (CD29, very late activation [VLA] members), beta 2 (leukocyte integrins such as CD11a/CD18, CD11b/CD18, CD11c/CD18, and alpha d beta 2), beta 3 (CD61, cytoadhesions), and beta 7 (alpha 4 beta 7 and alpha E beta 7). The immunoglobulin superfamily includes leukocyte function antigen-2 (LFA-2 or CD2), leukocyte function antigen-3 (LFA-3 or CD58), intercellular adhesion molecules (ICAMs), vascular adhesion molecule-1 (VCAM-1), platelet-endothelial cell adhesion molecule-1 (PE-CAM-1), and mucosal addressin cell adhesion molecule-1 (MAdCAM-1). The selectin family includes E-selectin (CD62E), P-selectin (CD62P), and L-selectin (CD62L). Cadherins are major cell-cell adhesion molecules and include epithelial (E), placental (P), and neural (N) subclasses. The binding sites (ligands/receptors) are different for each of these cell adhesion molecules (e.g., ICAM binds to CD11/CD18; VCAM-1 binds to VLA-4). The specific cell adhesion molecules and their ligands that may be involved in pathologic conditions and potential therapeutic strategies by modulating the expression of these molecules will be discussed.     

Increased expression of costimulatory molecules on peripheral blood monocytes in patients with Crohn's disease

BACKGROUND: Activation of T lymphocytes and monocytes/macrophages has been implicated in the pathogenesis of Crohn's disease (CD). Costimulatory molecules play important roles in optimal T-cell activation. METHODS: With flow cytometric analysis we have investigated the expression of the costimulatory molecules B7-1 (CD80), B7-2 (CD86), and CD18 and the intercellular adhesion molecule-1 (ICAM-1) on peripheral blood monocytes and the expression of the activation markers HLA-DR and IL-2R (CD25) on peripheral blood T lymphocytes from 31 CD patients, 17 ulcerative colitis (UC) patients, and 10 healthy controls. RESULTS: In CD patients the percentage of activated T cells (CD3+ HLA-DR+ and CD3+ IL-2R+) was significantly increased compared with those of controls and UC patients (P < 0.05). Most monocytes from all three groups expressed B7-2, CD18, and ICAM-1 molecules (all > 79%), but only a few positive cells expressed B7-1 molecules (< 5%). No significant differences were detected in the percentage positivity of all costimulatory molecules tested among CD, UC, and controls. The mean fluorescence intensity (MFI) of B7-1 in all three groups was very weak and not significantly different. However, in CD patients there was a significantly increased MFI of B7-2, CD18, and ICAM-1 molecules compared with UC and controls (P < 0.05). On the other hand, both the percentage positivity and MFI of HLA-DR molecules on monocytes of UC patients were significantly lower than those of CD patients and controls (P < 0.05). CONCLUSIONS: Expression of the costimulatory molecules B7-2, CD18, and ICAM-1 on peripheral blood monocytes of CD patients is increased. In CD patients activation of peripheral T lymphocytes may correlate with increased expression of these costimulatory molecules on peripheral blood monocytes.     

Expression of vascular adhesion molecules in saphenous vein coronary bypass grafts

BACKGROUND: Adhesion of blood elements to the endothelium is an important step in the development of vein graft disease. This study examines the expression of vascular adhesion molecules on explanted saphenous vein bypass grafts. METHODS: Immunocytochemical staining was performed using explanted saphenous vein grafts from 28 patients. Antibodies against the endothelial markers CD31, von Willebrand factor, intercellular adhesion molecule-1, vascular adhesion molecule-1, and E-selectin were used. RESULTS: Staining for CD31 and von Willebrand factor demonstrated the presence of endothelial cells in the lumen and the vasa vasorum. Expression of intercellular adhesion molecule-1 was variable between grafts, whereas vascular adhesion molecule-1 and E-selectin were almost always absent on the luminal endothelium. In contrast, the endothelium of the vasa vasorum stained positively for intercellular adhesion molecule-1 and vascular adhesion molecule-1, and was also seen on nonendothelial cells within the vessel wall. Expression of these adhesion molecules did not vary with the severity of vein graft disease. CONCLUSIONS: This study highlights the blood vessels in the adventitia as possible sites for the adhesion and migration of cells into the vessel wall.     

Increased expression of CD11b/CD18 on phagocytes in ischaemic disease: a bridge between inflammation and coagulation

The aim of this study was to assess the expression of CD11b/CD18 integrin adhesion molecules on the phagocytes of patients with ischaemic diseases, and to evaluate the concentration of soluble adhesion molecules that are released from endothelium (sICAM-1) and from phagocytes (sL-selectin). A total of 370 patients were enrolled: 120 with coronary artery disease (CAD); 50 with peripheral artery occlusive disease (PAOD); and 200 control subjects with no clinical manifestations of ischaemic disease. CD11b/CD18 integrin was detected by flow cytometry, whereas sL-selectin and sICAM-1 concentrations were detected using a sandwich-type immunoassay. CD11b/CD18 integrin expression was found to be higher in the patients with ischaemic disease than in the control subjects (P < 0.001). The PAOD patients had higher values of CD11b/CD18 integrin than the CAD ones (P < 0.01). The concentration of soluble adhesion molecules did not show any significant differences within the three groups (P = NS). The high expression of CD11b/CD18 integrin in ischaemic disease patients may depend on the increased, but probably stable, cytokine network that has been demonstrated to occur in chronic ischaemic diseases: the difference observed between PAOD and CAD patients could be the consequence of higher inflammatory activation probably resulting from the greater extent of the atherosclerotic process in PAOD, or of the more localized ischaemic area in CAD patients. CD11b/CD18 can therefore be considered a marker of chronic phagocyte activation during ischaemic disease. On the other hand, sICAM and sL-selectin concentrations were found to be within the normal range; they have recently been considered as a marker for acute ischaemic events and acute inflammatory process activation. Our results confirm that in uncomplicated atherosclerosis no acute inflammatory process activation should occur.     

Activation of peripheral blood T cells by interaction and migration through endothelium: role of lymphocyte function antigen-1/intercellular adhesion molecule-1 and interleukin-15

Cell adhesion molecules have a key role in the migration of T cells to inflammatory foci. However, the effect of the endothelial-lymphocyte interaction on the activation of the latter cells remains unresolved. We have studied the effect of resting and stimulated endothelial cells (ECs) on the activation of peripheral blood T cells (PBTLs), as assessed by the expression of CD69 and CD25 activation antigens. The incubation of PBTLs with tumor necrosis factor-alpha-activated EC monolayers, either alive or fixed, induced the expression of CD69 but not CD25, preferentially in the CD8(+) CD45RO+ cell subset. Furthermore, it induced the production of cytokines such as IFN-gamma, but not that of interleukin-2 (IL-2) and IL-4. EC treated with other stimuli such as IL-1beta, IFN-gamma, or lipopolysaccharide also showed the same proactivatory effect on T cells. Lymphocyte activation was almost completely inhibited by blocking anti-CD18 and anti-intercellular adhesion molecule-1 (anti-ICAM-1) monoclonal antibodies (MoAbs), but only slightly affected by MoAbs against CD49d, vascular cell adhesion molecule-1, and anti-IL-15. In addition, the interaction of PBTL with immobilized ICAM-1 induced CD69 expression in the same memory T-cell subset. IL-15 induced T-cell activation with expression of CD69 and CD25, and production of IFN-gamma, and its effect was additive with that triggered by cell adhesion to either EC or immobilized ICAM-1. The transmigration of PBTLs through either confluent EC monolayers or ICAM-1-coated membranes also induced efficiently the expression of CD69. When IL-15 was used as chemoattractant in these assays, a further enhancement in CD69 expression was observed in migrated cells. Together these results indicate that stimulated endothelium may have an important role in T-cell activation, through the lymphocyte function antigen-1/ICAM-1 pathway, and that IL-15 efficiently cooperates in this phenomenon. These observations could account for the abundance of CD69(+) cells in the lymphocytic infiltrates of several chronic inflammatory diseases.