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A correction has been published: N Engl J Med 1999;341(22):1708.
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Background The cause of persistent eosinophilia and the hypereosinophilic syndrome is unknown. Recent work suggests that in some patients with the hypereosinophilic syndrome, a clone of abnormal T cells produces large amounts of interleukin-5, a cytokine required for the growth and differentiation of eosinophils. We examined T-cell surface markers, rearranged T-cell–receptor genes, and in vitro production of cytokines by T cells from patients with idiopathic eosinophilia.
Methods The expression of surface molecules on T cells was measured by flow cytometry. Cytokine expression was measured by enzyme-linked immunosorbent assay, flow cytometry, and immunohistochemical analysis. To identify dominant (clonal) rearrangements of the T-cell receptor within the lymphocyte population, Southern blot analysis (ß chain) and the polymerase chain reaction (
Results Among 60 patients with idiopathic eosinophilia, 16 had circulating T cells with an aberrant immunophenotype. In each of these patients, the abnormal immunophenotype was unique. Evidence of clonal rearrangements of the T-cell receptor was obtained in 8 of the 16 patients. In most instances, the abnormal T cells expressed large amounts of surface proteins associated with T-cell activation (the
Conclusions Clonal populations of abnormal T cells producing interleukin-5 occur in some patients with idiopathic eosinophilia.
chain) were performed according to standard protocols. 
chain of the interleukin-2 receptor and the HLA-DR antigen). Moreover, the aberrant T cells produced large amounts of interleukin-5 in vitro.
We assessed 60 patients with idiopathic eosinophilia. Among this group, we identified 16 patients who had T cells with an abnormal immunophenotype. At the time of the initial blood analysis, 15 of these 16 patients had no evidence of a malignant lymphoproliferative disorder. The immunophenotypically abnormal T cells in the blood of these patients produced large amounts of interleukin-5.
Methods
Patients
A total of 59 adults and 1 child with idiopathic eosinophilia were evaluated during the period from June 1993 to December 1998. Immunologic analysis of blood leukocytes revealed a subgroup of 16 patients in whom T cells with an aberrant immunophenotype were detected.
Immunofluorescence Analysis
Peripheral-blood T lymphocytes were analyzed by standard flow-cytometric techniques. Initial screening for T cells with an aberrant immunophenotype was performed with a panel of monoclonal antibodies against the lineage-associated T-cell markers CD2, CD3, CD4, CD5, CD6, CD7, and CD8 in two-color immunofluorescence analysis (all antibodies were obtained from Coulter, Hialeah, Fla.).
The aberrant expression of at least one T-cell surface molecule led to further characterization of these cells. For this purpose, we used the monoclonal antibody that detected decreased or increased staining of the surface marker to identify the abnormal T cells. This antibody was used in combination with fluorescein- or phycoerythrin-conjugated monoclonal antibodies against activation-associated molecules (CD25, HLA-DR, and CD95) and fluorescein-conjugated monoclonal antibodies against several Vß families of the T-cell receptor. CD25 is the chain of the interleukin-2 receptor, HLA-DR is a family of class II HLA molecules, and CD95 (also called the Fas receptor or APO-1) is a receptor that induces programmed cell death (apoptosis) in activated T cells.
In additional experiments, staining for interleukin-5 in aberrant and CD25+ circulating T cells was performed with two-color fluorescence analysis,8 in which fluorescein- and phycoerythrin-conjugated anti-CD95 monoclonal antibodies were used (Immunotech, Marseilles, France). Surface expression of the Vß chains of the T-cell receptor was detected with the following fluorescein-conjugated monoclonal antibodies: anti-Vß2 and anti-Vß19 (Immunotech) and anti-Vß5a, anti-Vß5b, anti-Vß5c, anti-Vß6.7, anti-Vß8, and anti-Vß12 (T Cell Diagnostics, Cambridge, Mass.). Monoclonal antibodies against the /ß T-cell receptor and the /
T-cell receptor (TCR-1) were also used (T Cell Diagnostics), as were R-phycoerythrin–conjugated monoclonal antibodies against interleukin-5 (PharMingen, San Diego, Calif.).
To quantify the expression of lineage-associated surface markers, the mean channel fluorescence levels for each marker on the aberrant T cells and the remaining normal T cells in each patient were compared, and the ratios were calculated as the mean channel fluorescence levels for aberrant T cells divided by the mean channel fluorescence levels for normal T cells. A value of 1.0 indicates normal expression of the marker, a value less than 1.0 indicates decreased expression, and a value greater than 1.0 indicates increased expression.
To quantify the expression of activation-associated surface markers on aberrant T cells, we identified the cells with a monoclonal antibody against a lineage-associated molecule, as described above. The mean channel fluorescence level for each activation-associated marker was compared with that for the isotype-matched control monoclonal antibody for each patient, and the ratios were calculated as the mean channel fluorescence level for an activation-associated molecule divided by the mean channel fluorescence level for control antibody staining. A value of 1.0 indicates no expression, and a value greater than 1.0 indicates expression. Since increased T-cell activation has been reported in patients with asthma,9 we determined the relative expression of CD25, HLA-DR, and CD95 in peripheral-blood CD4+ T cells from 10 patients with bronchial asthma for comparison with the patients with eosinophilia.
Rearrangements of the T-Cell–Receptor Gene
Southern blot analysis of the gene coding for the ß chain of the T-cell receptor was carried out as previously described.7 To identify clonal rearrangements of the chain of the T-cell receptor, we used the polymerase chain reaction (PCR), followed by electrophoresis on a denaturing gel with a formamide–urea gradient.10 The PCR method incorporated primers for the variable regions 1 to 9, which detect approximately 90 percent of all rearrangements of the T-cell–receptor chain. For both Southern blot analysis and the PCR method, DNA preparations from peripheral-blood mononuclear cells were used. Additional DNA was extracted from purified CD3– T cells with low amounts of CD4 (Patient 7) and biopsy specimens taken from the intestine (Patient 2) and skin (Patients 1, 4, 5, 11, 13, and 15).
Expression of Cytokine Genes
Interleukin-5 messenger RNA (mRNA) and protein were measured in peripheral-blood mononuclear cells. Interleukin-5 mRNA was detected by reverse-transcription PCR (RT-PCR),2,7 and interleukin-5 protein levels were quantified in supernatants from peripheral-blood mononuclear cells by enzyme-linked immunosorbent assay after a 24-hour incubation period without any in vitro activation.7 Levels of interleukin-4 and interferon- were also measured in these supernatants. Interleukin-5 immunoreactivity was determined in tissue sections by immunohistochemistry with the use of the alkaline phosphatase–anti-alkaline phosphatase technique.2,11
Results
Patients
The clinical and hematologic features of the 16 patients we studied are shown in Table 1 and Table 2. Only one patient presented with lymphocytosis, and he was subsequently given a diagnosis of the Sézary syndrome. Ten of the patients met the diagnostic criteria for the hypereosinophilic syndrome.4 The remaining six patients had eosinophilia of unknown cause, but their conditions did not meet all the criteria for the diagnosis of the hypereosinophilic syndrome. None of the 16 patients had detectable parasitic infection or human immunodeficiency virus infection. Cardiac complications were not observed. Eight patients had elevated serum IgE levels, but only Patient 1 had an IgE-mediated allergic disease (allergic asthma).
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Immunophenotypes of T Cells
Among the 60 patients with idiopathic eosinophilia, 16 had circulating T cells with an abnormal immunophenotype (Table 3). In 10 of these 16 patients, the ratio of CD4 cells to CD8 cells was decreased or increased (Table 2). Direct evidence of the presence of aberrant T cells was obtained by two-color immunofluorescence assays (Figure 1). Each patient had a unique subgroup of T cells with an aberrant immunophenotype (Table 3). Nine patients (Patients 1, 4, 6, 9, 10, 12, 13, 14, and 15) had abnormal CD3+CD4+CD8– T cells, three patients (Patients 5, 8, and 16) had abnormal CD3+CD4–CD8+ cells, and three patients (Patients 2, 11, and 15) had abnormal CD3+CD4–CD8– cells (Table 3). CD3–CD4+ T cells were found in two patients (Patients 3 and 7). Patient 15 had two populations of aberrant T cells. In 13 patients, abnormal expression of the lineage-associated T-cell markers CD2, CD5, CD6, and CD7 contributed to the overall aberrant immunophenotype. Loss of expression as well as decreased or increased amounts of surface proteins was found in individual patients (Figure 1 and Table 3). Excluding Patient 6, who had an absolute lymphocyte count of almost 9000 per cubic millimeter, the proportion of abnormal T cells in the remaining 15 patients ranged from 6.5 percent to 55.8 percent of all lymphocytes. In the patient with lymphocytosis, 70.0 percent of the lymphocytes had an aberrant immunophenotype (CD2–CD3+).
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chain of the interleukin-2 receptor), HLA-DR molecules, or both. The expression of CD25 and HLA-DR by these cells was often extremely high as compared with the peripheral-blood CD4+ T cells of patients with bronchial asthma (Figure 2B and Table 3). Another abnormality was the absence of CD95, the Fas receptor involved in apoptosis, in the aberrant T cells of 8 of 15 patients. In three patients (Patients 4, 9, and 14), we identified a population of monoclonal T cells by flow cytometry; all the T cells with an aberrant immunophenotype in these patients expressed one particular family of Vß genes (Figure 1F and Table 3).
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-chain genes of the T-cell receptor by Southern blotting or PCR analysis or both (Table 3). In the remaining eight patients, these molecular studies failed to detect monoclonality. Nevertheless, the aberrant T cells of these patients did not bind eight monoclonal antibodies against different Vß families, suggesting the possibility of oligoclonal populations.
Patient 9 was studied for more than 13 years. During the first two years after the onset of the disease, as ascertained by the diagnosis of pruritic poikiloderma, the number of eosinophils and the ratio of CD4 T cells to CD8 T cells in peripheral blood were within the normal range. Two years later, the CD4:CD8 ratio was 7.7 and the number of peripheral-blood eosinophils was 3082 per cubic millimeter but remained constant for more than nine years. A PCR-based test for the presence of clonal rearrangements of the chain of the T-cell receptor, performed eight years after the initial skin symptoms appeared, was negative. After five more years, a monoclonal T-cell population was detected by flow cytometry.
In Patient 14, therapy with interferon alfa-2b (Intron, Essex, Munich, Germany) was associated with the disappearance of the atypical clonal T cells within a few weeks. The patient had no evidence of circulating aberrant T cells and had low numbers of eosinophils for about nine months. Then, because of an exacerbation of skin symptoms, the treatment with interferon alfa-2b was stopped. Circulating T cells with the same aberrant immunophenotype reappeared, and malignant T-cell lymphoma was subsequently diagnosed on the basis of a lymph-node biopsy. The transition to malignant T-cell lymphoma was also observed in Patients 2 and 12 and was associated with a rapid increase in the numbers of leukocytes, lymphocytes, abnormal T cells, and eosinophils in peripheral blood. In these three patients, the lymphoma cells had the same immunophenotype as the clonal subpopulation of T cells in the blood.
Production of Interleukin-5
In vitro, unstimulated peripheral-blood mononuclear cells from 12 of 14 patients tested released detectable amounts of interleukin-5 into the supernatant within a 24-hour incubation period (Table 4). In addition, interleukin-5 mRNA was detected in these cells by RT-PCR (data not shown). Interleukin-4, interferon-, or both were found in the supernatants of cultured peripheral-blood mononuclear cells in six patients (Table 4). Unstimulated peripheral-blood mononuclear cells from control subjects did not release measurable amounts of interleukin-4, interleukin-5, or interferon- (data not shown). As shown in Figure 2C, 2D, and 2E, monoclonal populations of T cells with high levels of expression of CD25 from Patients 10, 14, and 15 contained high amounts of cytoplasmic interleukin-5, as assessed by flow cytometry. Clonal T cells from Patients 2 and 3 were purified and then cultured. We found high levels of interleukin-5 in supernatants of these cultures (data not shown); T cells with an aberrant immunophenotype were also identified in tissues and found to express interleukin-5 (Figure 3).
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In this study of 60 patients with idiopathic eosinophilia (median number of eosinophils, 3051 per cubic millimeter), we identified 16 patients with abnormal T cells in peripheral blood. Cultures of the peripheral-blood mononuclear cells of 12 patients revealed detectable amounts of interleukin-5 in the supernatant, and the abnormal T-cell populations of 7 of these patients were directly shown to express high levels of interleukin-5. It is likely that the abnormal T cells producing interleukin-5 caused the eosinophilia in these 16 patients.
The abnormal T cells in the 16 patients were identified by studies of their surface markers. In each case, the constellation of abnormalities of these markers was unique, but in 14 patients the aberrant T cells had the immunophenotype of activated T cells. The abnormal T cells constituted 6.5 percent to 55.8 percent of the circulating lymphocytes, and in eight patients they had the features of a clonal population. Also in eight patients, the aberrant T cells lacked detectable receptors for the Fas ligand (CD95), which is a mediator of apoptosis. It is likely that CD95-mediated apoptosis, which is essential for homeostasis of lymphocytes,13 does not function in these patients. Treatment with low-dose interferon-alfa-2b might induce apoptosis in T cells that lack CD95.14,15,16
It was not possible to demonstrate the presence of clonal T cells in eight patients by molecular techniques, but the presence of an aberrant T-cell immunophenotype implied the presence of a clone. These observations concur with previous work in which clonal rearrangements of the variable-region genes of the T-cell receptor were not found in some well-documented cases of T-cell lymphoma.17,18 Our PCR technique may not have covered all possible clonal -chain rearrangements of variable regions 10 and 12. Another possibility is that the limited sensitivity of the technique precludes the demonstration of a monoclonal cell population in all cases.18 The lack of detectable clonal rearrangements of T-cell receptors does not rule out the possibility of a premalignant or neoplastic clone of T cells. As we observed in Patient 3, the detection of a clonal gene rearrangement was possible only several years after the identification of peripheral-blood T cells with an aberrant immunophenotype.
Eight patients had some clinical features of the Sézary syndrome, including pruritic scaling erythroderma.19,20 Indeed, Patient 6 was ultimately given a diagnosis of the Sézary syndrome. However, in all other patients, this diagnosis was ruled out. In our study, we did not observe any CD4+CD7–CD25– T cells, which are typical of cells in patients with the Sézary syndrome.21,22
Our results indicate that clonal populations of T cells with an abnormal immunophenotype that secrete interleukin-5 are present in some patients with idiopathic eosinophilia. It is unknown whether these cells arise in reaction to chronic inflammation (e.g., inflammatory skin disease) or represent the precursors of neoplastic T cells that secrete abnormally high amounts of interleukin-5 (thereby causing eosinophilia). Nevertheless, it may not be coincidental that in 4 of the 16 patients we studied, T-cell lymphoma or the Sézary syndrome was ultimately diagnosed. The fact that the lymphoma cells in three patients had the same immunophenotype as the clonal population of T cells in the blood supports the idea that abnormal T cells in patients with persistent eosinophilia can be the precursors of malignant T cells.
Supported by grants from the Swiss National Science Foundation (32-49210.96), the Saurer Foundation, the OPO-Foundation, and the Foundation for Scientific Research of the University of Zurich.
We are indebted to the many clinicians who allowed us to analyze their patients and provided clinical information; to Drs. D.R. Zimmermann and M. Kleinhans (University of Zurich, Zurich, Switzerland), who did most of the studies of T-cell–receptor rearrangements; to Martina Weber, Ivo Balatti, and Beate Rückert (Swiss Institute of Allergy and Asthma Research, Davos, Switzerland) for their excellent technical assistance; to Dr. Stephanie Gratzl (Swiss Institute of Allergy and Asthma Research), who was involved with the immunohistochemical studies; to Dr. J. Tavernier (University of Ghent, Ghent, Belgium) for providing anti–interleukin-5 monoclonal antibodies; and to Dr. M. Goldman (University of Brussels, Brussels, Belgium) for critical reading and discussion of the manuscript.
Source Information
From the Swiss Institute of Allergy and Asthma Research, University of Zurich, Davos, Switzerland (H.-U.S., K.B.); the Research Center for Environment and Health, Technical University of Munich, Munich, Germany (S.G.P.); and the Department of Dermatology, University of Zurich, Zurich, Switzerland (R.D.).
Address reprint requests to Dr. Simon at the Swiss Institute of Allergy and Asthma Research, University of Zurich, Obere Strasse 22, CH-7270 Davos, Switzerland, or at hus{at}siaf.unizh.ch.
References
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Related Letters:
Idiopathic Eosinophilia
Guitart J., Roufosse F., Schandené L., Cogan E., Suzuki R., Seto M., Nakaura S., Simon H.-U., Dummer R.
Extract |
Full Text
N Engl J Med 2000;
342:659-661, Mar 2, 2000.
Correspondence
This article has been cited by other articles:
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