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Original Studies |
University of Vienna, Vienna, Austria
Address all correspondence and requests for reprints to: Alois Gessl, M.D., Division of Endocrinology and Metabolism, Department of Medicine III, University of Vienna, Währinger Gürtel 18–20, 1090 Vienna, Austria. E-mail: alois.gessl{at}akh-wien.ac.at
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Abstract |
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-chain (CD25), and CD69 by three-color flow
cytometry. Twenty-six healthy subjects (HS; age, 32.0 ± 8.2 yr)
served as controls. No deviation was seen in either IDDM group compared to HS in CD25 expression on CD4+ or CD8+ cells or in their CD45RA+ or CD45RA- subsets. HLA-DR expression, however, was increased (P < 0.05) in total CD8+ cells and CD45RA+ cells, with CD45RA- CD8+ cells joining the prevailing pattern only in DML. Among CD4+ cells, increased expression of HLA-DR molecules was restricted to total and CD45RA- cells in DML. CD69 expression did not differ between IDDM and HS, but differed between DML (CD4+, CD8+, and CD45RA- CD4+) and DM only.
In conclusion, our data demonstrate that HLA-DR expression in IDDM is restricted to memory cells (CD45RA-) among CD4+ cells in DML and is more markedly confined to naive (CD45RA+) than to memory CD8+ cells, whereas the early activation antigen CD69 is more readily expressed in DML than in DM. The observed activation of circulating T cells suggests an ongoing immune process in IDDM both at clinical manifestation and after long duration.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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By analysis of CD45RA isoforms the reciprocal subpopulations of CD45RA+ CD45R0- (naive, unprimed) and CD45RA- CD45R0+ (memory, primed) may be differentiated among CD4+ and CD8+ T cells (16, 20, 21, 22, 23, 24). In IDDM of recent onset (DM), the proportions of CD45RA+ cells in the CD4+ T cell subset have been described to be normal (7, 12, 25), decreased (4), or increased (14), with the discrepancy also applying to prediabetic individuals [increased (7), decreased (26)]. Recently, a markedly increased proportion of CD45RA+ CD4+ and CD8+ cells carrying an activated phenotype has been observed in autoimmune thyroid disease (AITD) (23, 24). Increased proportions of CD45RA+ HLA-DR+ CD8+ cells have subsequently also been reported in patients with IDDM (27, 28).
The role of such CD45RA+-activated lymphocyte subpopulations in the pathogenesis of endocrine autoimmune disorders is unclear, but may reflect both an ongoing autoimmune process as well as a secondary immune phenomenon (5, 18).
As the immune process in IDDM appears to start many months before its clinical manifestation (29), persisting for years in those diabetic subjects manifesting in adulthood (30), we set out to define in more detail specific abnormalities of T cell subsets in IDDM of long standing (DML; median duration of disease, 8.7 yr) and in DM by identifying circulating activated (CD25+, HLA-DR+) CD4+ and CD8+ cells and their respective CD45RA+ and CD45RA- subsets. In addition, membrane expression of CD69, a member of the C-type animal lectin superfamily of signal transmitting receptors, was determined, reflecting early T cell activation (31, 32).
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Thirty-one consecutive patients with DM (13 women and 18 men; median age ± SD, 28 ± 6.9 yr; median hemoglobin A1c, 12.7 ± 2.6), classified according to WHO criteria (33) and 27 patients with DML receiving intensified (functional) insulin therapy (34) (12 women and 15 men; 33 ± 10.4 yr; median duration of disease, 104 ± 86 months; median hemoglobin A1c, 6.8 ± 0.7) were included in the study. DM patients had been receiving insulin treatment for less than 7 days and were studied after their admission to the hospital and stabilization of metabolic control to mean blood glucose levels below 140 mg/dL at a minimum of 4 blood glucose measurements/day. Apart from 2 individuals (mild retinopathy), DML patients did not display any late diabetes-associated complications, as determined by standard fundoscopy after pupil dilatation, and all had normal renal function (negative microalbuminuria, normal blood urea nitrogen, creatinine, and urinalysis). Twenty-six healthy subjects (HS: 15 women and 11 men) of similar age (median, 32 ± 8.2 yr; P > 0.05 vs. DM and DML) served as controls and were recruited from laboratory staff and blood donors during the same time period. They had no family history of IDDM and no clinical signs or symptoms of intercurrent illness. None of the patients or subjects had a family history of autoimmune endocrine disease.
Monoclonal antibodies (mAbs)
Fluorescein isothiocyanate-, phycoerythrin-, or peridinin chlorophyll protein-labeled mAbs were obtained from Becton Dickinson (Becton Dickinson Immunocytometry Systems, San Jose, CA) as follows: CD19, CD3, CD4, CD8, CD25, HLA-DR, CD69, CD45/CD14 (Leukogate), and isotype-specific fluorochrome-matched control mAbs. Fluorescein isothiocyanate-labeled mAb 2H4, recognizing the CD45RA isoform of the common leukocyte antigen (22), was purchased from Coulter Immunology (Hialeah, FL).
Lymphocyte phenotyping
Flow cytometry was performed using a lysed whole blood method (23, 24) to avoid possible artifactual changes in lymphocyte subpopulations (35). Briefly, 100 µL ethylenediamine tetraacetate whole blood were incubated for 30 min in the dark at room temperature with the appropriate dilution of mAbs, lysed for 5 min with FACS lysing solution (Becton Dickinson), then washed twice with Hanks’ Balanced Salt Solution containing 0.2% BSA and 0.1% sodium azide. Flow cytometry was performed on a FACScan using Lysis software (Becton Dickinson). A lymphocyte gate was set using CD45 and CD14 to backgate on the scatter plot. Background fluorescence was assessed with the appropriate isotype- and fluorochrome-matched control mAbs directed against an irrelevant target to allow determination of the percentage of positive cells. Data from at least 104 CD4+ or CD8+ cells were analyzed.
Statistical analysis
Data are expressed as scatter plots and medians in the figures
and as the mean ± SD in the text, unless stated
otherwise. After square root transformation, ANOVA (that gave a
P < 0.0001 for all parameters) followed by Tukey’s
Studentized range test for pairwise comparisons was used to test for
group differences at a significance level of 95%, setting
P < 0.05 as indicated by brackets in
Figs. 2–5
.
Regression and correlation analyses were employed as appropriate.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The proportion of lymphocyte subpopulations did not display any differences between patients with DM or DML and HS. In particular, no differences were seen in levels of CD4+ and CD8+ cells (not shown).
Analysis for CD4+ and
CD8+ subsets (Fig. 1, A and B)
The mean percentages of CD45RA+ cells among
CD4+ (DM, 41.2 ± 12.9%; DML, 40.9 ± 12.5%;
HS, 47.5 ± 16.6%; P = NS) and CD8+
(64.5 ± 15.2% and 63.9 ± 12.7%; HS, 72.2 ± 12.1%;
P = NS) cells were somewhat smaller in both IDDM groups
than in HS. For all groups studied, an exponential relationship was
apparent between the fraction of CD4+ and CD8+
cells carrying the CD45RA isoforms (Fig. 1C; HS, r = 0.85; DM,
r = 0.65; DML, r = 0.70; all subjects, r = 0.74).
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No deviation was seen in CD4+ or CD8+ cells in patients with DM or DML vs. HS in the expression of CD25 in the total population as well as in their CD45RA+ and CD45RA- subsets (not shown).
HLA-DR expression on T cells
The mean percentage of HLA-DR expression on CD4+ cells
was 1.3-fold greater in DML than in HS (P < 0.05), but
not in DM (1.14-fold; P = NS; Fig. 2A
). This rise in the HLA-DR+
CD4+ T cell population was mainly due to HLA-DR expression
among CD45RA- (+32%; P < 0.05; Fig. 2B),
but not CD45RA+ CD4+, cells (+19%;
P = NS; Fig. 2C).
The mean percentage of HLA-DR+ cells among CD8+
cells was increased compared to that in HS in both DM (+39%;
P < 0.05) and DML (+57%; P < 0.05;
Fig. 3A) as well as in the respective
CD45RA+ subset (DM, +45%; DML, +76%; P <
0.05; Fig. 3C). HLA-DR epitopes were significantly elevated among
CD45RA- CD8+ cells in DML (+40%;
P < 0.05), but not in DM (+34%; P =
NS), compared to those in HS (Fig. 3B).
CD69 expression on T cells
None of the diabetic subjects differed from the group of HS in the
percentage of CD69+ cells among CD4+ (Fig. 4, A–C) and CD8+ (Fig. 5, A–C) cells and their subsets.
However, patients with DML had a significantly greater percentage of
CD69 T cells among both total CD4+ (+48%;
P < 0.05; Fig. 4A) and CD8+ (+40%;
P < 0.05; Fig. 5A) cells and also in the
CD45RA- compartment of CD4+ cells (+50%;
P < 0.05; Fig. 4B) compared with DM.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The greater expression of HLA-DR on CD8+ both in DM (+39%) and DML (+57%) vs. HS is in line with previous data on the behavior of CD3+ (5, 6, 19) and CD8+ T cells [DM (8, 19), DML (5, 13, 19)]. The same applies to the observation of more marked HLA-DR expression on CD4+ cells in DML, which seems to be restricted to memory cells, whereas at manifestation of IDDM it occurs in both memory (27) and naive T cells (27, 28). HLA-DR expression was, however, not restricted to the CD45RA- compartment (21) of CD8+ cells in DM, but, rather, favored the CD45RA+ subset. This is at variance with the observation of an equally increased HLA-DR expression on both CD8+ cell subsets in IDDM at manifestation (27). The cause of these discrepancies may stem from a too small number of subjects investigated as well as the previous use of less developed detection techniques and indirect immunofluorescence (28). Furthermore, lymphocyte enrichment by Ficoll density gradient before staining and analysis may affect lymphocyte subset composition (9, 35). The observed deviations may also relate in part to the different distribution in age, which is known to reduce the expression of CD45RA (36). As in our study age was similar in DML and HS, such variation has not contributed to differences in CD45 expression.
In addition, patients with DM and DML did not differ from HS in the proportion of CD4+ and CD8+ lymphocytes in peripheral blood, which deviates from findings in patients with AITD (23) and some reports in IDDM (13, 14, 37, 38). No significant difference in expression was observed in the CD45RA isoform on CD4+ and CD8+ cells between IDDM and HS, which is in line with some (4, 7, 14, 28), but not all (12, 38), previous reports. In contrast to earlier observations (4, 11, 12, 17), this study describes expression of CD25 on circulating T cells to be identical in IDDM and in HS both upon bulk and subpopulation (CD45RA+ and CD45RA-) analyses. The report of CD4+ CD25+, but not CD3+ CD25+ and CD8+ CD25+, cells to be slightly increased in prediabetic and nondiabetic twins vs. control subjects is subject to doubt, as previous data were reported as a percentage of all cells in the lymphocyte gate (18) and thus were influenced by relative changes in B cells, natural killer cells, and T cells.
Comparing patients suffering from AITD with those with IDDM, the latter shared increased HLA-DR expression on CD45RA+ CD8+ cells, but lacked the increased CD25 expression observed on CD45RA+ CD4+ cells (23, 24). These differences might be attributable to the different underlying metabolic abnormalities and initiating events. However, as activated CD45RA+ T cells have been also seen in patients with rheumatoid arthritis (39), their presence could be a more general feature of autoimmune disease.
The reason for ongoing lymphocyte activation, as judged from HLA-DR and CD69 expression in DML, is unknown. As only patients with excellent metabolic control and virtually without clinically manifest late complications were studied, an effect of chronic hyperglycemia or the presence of chronic inflammation or late diabetic complications can be ruled out. In that context it is of note that metabolic state does not affect the prevalence of lymphocyte subsets (40). Therefore, persistent lymphocyte activation may reflect an ongoing autoimmune process, as augmented expression of HLA-DR and CD69 on CD4+ and CD8+ T cells suggests activation of a cellular immune response in IDDM both at its onset and during the course of the disease. In addition, our finding of a more marked activation of CD69+ T cells in DML vs. DM is in line with data suggesting partially down-regulated T cell activation at manifestation of disease (albeit partially due to metabolic abnormalities and stress associated with acute illness) and vanishing with its prolonged duration (16, 41). As CD69 expression depends on several signal transducing pathways (42) that are impaired in newly diagnosed IDDM (16, 41), higher CD69 expression in DML than in DM hints at a fading of such inhibition with prolonged duration of the disease. This lack of inhibition could also contribute to increased HLA-DR expression in DML.
These phenomena are markedly restricted to the compartment of
CD8+ T cells and are in line with the finding in recently
diagnosed IDDMs of activated CD8+ cells predominating in
pancreatic tissue infiltrates as well as the increased HLA-DR
expression on circulating cells (43). These cells have a phenotype of
cytotoxic (CD11b-) T cells (28). A role of
CD8+ cells is also suggested by islet hyperexpression of
class I major histocompatibility complex molecules, mediating target
recognition by CD8+ T cells (3). In NOD (nonobese diabetic)
mice, a probable pathogenic role of naive (CD45RA+) T cells
has been suggested, as CD45RA+ CD4+ cells
predominated in early islet infiltrates (44). In addition, the
gut-specific homing receptor
4ß7-integrin expressed by glutamic
acid decarboxylase-specific peripheral blood lymphocytes (45) is
predominantly expressed by CD45RA+ cells (46). In that
context it remains, however, to be determined whether circulating
activated CD8+ cells reflect the true nature of the
inflammatory reaction in and around islet cells in IDDM.
As only patients manifesting IDDM in adulthood were included in this study, it cannot be excluded that remnants of ß-cells may still be present even years after manifestation, driving the autoimmune process. However, as presumably only a small part of activated CD8+ cells is targeted against ß-cell autoantigens, activation of the majority of circulating T cells may be a secondary phenomenon, depending on cytokine stimulation (47), that is still ongoing years after manifestation.
In summary, contrary to previous reports (4, 17), no increase or difference in CD25 expression was seen in IDDM vs. HS on either CD4+ or CD8+ T cells. However, it is demonstrated that IDDM patients (both DM and DML) have a greater proportion of major histocompatibility complex class II-expressing CD8+ cells than HS. In parallel, expression of CD69 was unchanged vs. that in HS, but was increased in DML vs. DM. Enhanced expression of HLA-DR on T cells was primarily on naive CD8+ T lymphocytes, suggesting a distinctive role in the pathogenesis of IDDM, making HLA-DR+ CD8+ cells a potential early diagnostic marker of an ongoing immune phenomenon. As increased HLA-DR expression is also found in prediabetic individuals (9, 10, 18), our findings may extend to such subjects and thus potentially help to identify subjects at risk for IDDM.
We conclude that the persistent expression of activation markers CD69 and HLA-DR, but not of CD25, on circulating CD8+ and CD4+ cells in patients with DML is indicative of an ongoing immune process. To better understand the roles of these activated T cell subsets in IDDM, a definition of their derivations, functions, and antigen specificities is urgently needed.
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Acknowledgments |
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Received October 23, 1997.
Revised February 24, 1998.
Accepted March 9, 1998.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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4ß7, on human
leukocytes. J Immunol. 153:517–528.[Abstract]
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