Log in using OpenID

A Case of Hypereosinophilic Syndrome Is Associated With - Blood

From by guest on December 22, 2014. For personal use only.
A Case of Hypereosinophilic Syndrome Is Associated With the Expansion of
a CD3-CD4+ T-cell Population Able to Secrete Large Amounts
of Interleukin-5
By Duilio Brugnoni, Paolo Airb, Giuseppe Rossi, Aiessandra Bettinardi, Hans-Uwe Simon, Lucia Garza,
Cinzia Tosoni, Roberto Cattaneo, Kurt Blaser, and Alessandra Tucci
Interleukin-5 (IL-5)
is the major soluble factor able
to mediate
syndrome in which the presence
CD3-CD4+ cells able to overproduce 11-5 was shown. The
lack of CD3 and TCRAB membrane expression on otherwise
phenotypically normal mature T lymphocytes together
the absenceofdetectableTCRBVmRNAandclonalrearrangementofTCRBgenesuggested
that the abnormal
lymphocyte population was the expression of a peripheral
T-cell lymphomawith an indolent clinical course. Peripheral
blood lymphocytes enriched in this population were able
but not ofIL-4,andnoIL-2or
interferon-y, when stimulated
in vitro with phytohemagglutinin and phorbol myristate acetate. The serum contained
eosinophil survival factors whoseactivity was partially
neutralized by a specific antihuman IL-5 antibody. This observation further emphasized
the relationship between hypereosinophilic
0 1996 by The American Society of Hematology.
s h ~ w n . ' ~Recently,
a case of clonal proliferation of Th2
cells in a man with the hypereosinophilic syndrome (HES)
was reported. The investigators showed the expansion of a
T-cell clone with the unusual phenotype CD3-CD4' able to
produce high levels of L - 4 and IL-5 and low levels of IL2 and IFW-7."
We report a similar case of HES in which the presence
of a population of CD3-CD4+ cells able to overproduce IL5 (but not IL-4) was shown.
HE RELATIONSHIP between hypereosinophilia and T
lymphocytes is well established' and is mediated by
the production of eosinophilopoietic cytokines. CD4+ lymphocytes are the most important source of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3
(IL-3), and IL-5, three currently recognized cytokines active
in promoting the development and differentiation of eosinophils in bone marrow.z In mice, T-cell clones can be classified according to the pattern of cytokine secretion and there
is good evidence that also human cells could be subdivided
into these two profile^.^ Type 1 helper T-cell (Thl) clones
secrete IL-2 and interferon-y (IFN-y), whereas type 2 (Th2)
clones secrete IL-4 and IL-5; both secrete IL-3 and GMCSF. Interestingly, these latter two cytokines also stimulate
the development of other marrow-derived cells, whereas IL5 in humans acts more specifically on eosinophils and is
the major, and possibly the only, cytokine involved in the
production of specific e~sinophilia.'.~
Over the last few years
it became evident that the IL-5 production plays a pivotal
role in the development of hypereosinophilia both in the
secondary forms associated with allergic diseases or parasite
infections' and in the idiopathic
at least in vitro, these cytokines dramatically increase the
life span of eosinophils by inhibiting their apoptotic cell
The association between hypereosinophilia and T-cell
lymphoma is also well established,""* and in some studies
the production of IL-5 by neoplastic T cells has been
From the Servizio di lmmunologia Clinica, CONBIOTEC, I I P
Laboratorio Analisi, Sezione di Ematologia, Spedali Civili di Brescia, Brescia, Italy; and the Swiss Institut of Allergy and Asthma
Research, Davos, Switzerland.
Submitted April 21, 1995; accepted September 14, 1995.
Supported in part by Consiglio Nazionale delle Ricerche (Grant
No. 94.01328.PF39).
Address reprint requests to Paolo Airo, MD, Servizio di Immunologia Clinica, Spedali Civili, 25123 Brescia, Italy.
The publication costsof this article weredefrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C.section 1734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
Case report. A 70-year-old man with hypereosinophilia for the
last 5 years has been investigated. His absolute eosinophil count
ranged from 2,700 to16,60O/pL.He
complained of generalized
pruritus with periodic eruptions of papular elements that on histologic examination showed nonspecific follicular inflammation. A
physical examination showed mild axillary and inguinal lymph node
enlargement and a histologic examination showed a reactive
lymphoid hyperplasia with marked infiltration of eosinophils and
polyclonal plasmacells with no evidence of lymphoma. Polyclonal
hypergammaglobulinemia ( y globulins: 41% of 9.6 g/dL of total
serum proteins) was present with high IgG and IgM and normal IgA
and IgE levels. Other laboratory parameters were normal as well as
peripheral blood eosinophil morphology.
Bone marrowbiopsy results showed marked eosinophilia and
moderate polyclonal plasmacytosis. Cytogenetic analysis showed a
normal karyotype. Causes of secondary eosinophilia were ruled out,
including allergic, parasitic, neoplastic, or autoimmune disorders,
and a diagnosis of HES was made. Complications of longstanding
hypereosinophilia, particularly cardiac, were not found, despite periodic clinical and echographic monitoring.
Surprisingly, despite a normal blood lymphocyte count, immunophenotypical studies showed that 38% of lymphocytes were
The patient was treated with 6 methylprednisolone (6MP; 16 mg/
d). After 1 month, cutaneous symptoms disappeared and the eosinophil level decreased to 177/pL. He is currently on chronic low-dose
6MP (20 mg/wk). The eosinophil level is in the range of 2,000 to
3,00O/pL. The CD3-CD4' population decreased from 38% to 33%
after 1 month and is now 13% to 17%.
Cell preparation. Mononuclear cells from the patient and from
an age-matched healthy volunteer with no history of allergy and
normal lymphocyte populations were separated from the peripheral
blood by Ficoll-Hypaque density gradient centrifugation and washed
three times in RPMI-1640 (GIBCO Laboratories, Grand Island, NY).
Depletion of CD3' cells from peripheral blood mononuclear cells
(PBMC) was obtained using negative selection with mouse IgGBlood, Vol 87, No 4 (February 15), 1996: pp 1416-1422
From by guest on December 22, 2014. For personal use only.
conjugated magnetic beads (Dynal, Oslo, Norway) precoated for 30
minutes at 4В°C with CD3 monoclonal antibody (MoAb; OKT3; Ortho Diagnostic, Raritan, NJ), as recommended by the manufacturer;
less than 4% of negatively selected PBMC expressed CD3 after
this procedure, as assessed by immunofluorescence analysis. This
population was enriched in CD3-CD4' cells (57%) and was therefore used in experiments of cytokine production as representative of
the unusual population. For TCRBV mRNA analysis, CD4TD3cells were further purified from CD3-depleted PBMC by positive
selection with CD4-coated beads (Dynal); more than 92% of positively selected cells were CD4+CD3-. For these experiments, CD3+
cells, purified by positive selection (96% CD3+, 61% CD4+, and
36% CDS'), were used as representative of a normal T-cell population.
Cell cultures. All experiments were performed in complete medium supplemented with 10% heat-inactivated fetal calf serum (FCS)
1% glutamine, and antibiotics. Proliferative responses were measured in triplicates of 200 pL containing l X lo5 total or CD3depleted PBMC using various combinations of stimulating agents:
immobilized anti-CD3 MoAb (60 ng/mL; OKT3); mitogenic combinationof anti-CD2 MoAbs 0KT1lz and OKTll,(l:lOOO; kindly
provided by Dr F. Malavasi, University of Turin, Turin, Italy); 1.2
pg/mL phytohemoagglutinin (PHA; Irvine Scientific, Santa Ana,
CA), 0.6 pg/mL pokeweed mitogen (PWM; GIBCO); 5 ng/mL phorbo1 myristate acetate (PMA; Sigma Labs, St Louis, MO) plus ionomycin (500 ng/mL; Sigma Labs). After 72 hours of culture at 37В°C.
5% CO,, the cells were pulsed overnight with 1 pCi ('H)-thymidine
and were harvested subsequently. Incorporated radioactivity was
measured by liquid scintillation counting (Beckman, Fullerton, CA)
and the results were expressed as the mean cpm of triplicates.
For cytokine production, 5 X lo5 total or CD3-depleted PBMC
were cultured at 37"C, 5% CO2 in 24-well flat-bottomed plates in a
final volume of 1 mL with or without PHA (1.2 pg/mL) or PHA
plus PMA (2 ng/mL). After 72 hours, supernatants were collected
and frozen at -20В°C.
Immunofluorescence. Evaluation of lymphocyte surface membrane expression was performed on heparinized whole blood samples
with the following MoAbs: fluorescein isothiocyanate (FITC)-conjugated OKT3 (CD3), FITC-OKT4 (CD4), FITC-OKT8 (CDS), FITCOKTl1 (CD2), phycoerythrin (PE)-conjugated OKDR (HLA DR),
OKMl (CD1 lb) from Ortho, FITC-Leu4 (CD3), FITC-TCR alpha/
beta (TCRAB), PE-Leu 28 (CD28). PE-Leu 45RO (CD45RO), PEIL2R (CD25). PE-Leu1 IC(CD16), Leu 9 (CD7), Leu 7 (CD57), Leu23 (CD69) from Becton Dickinson (Mountain View, CA), FITC-B4
(CD19), PE-2H4 (CD45RA), PE-TI (CD5) from Coulter Immunology (Hialeah, FL), FITC-TCR Delta 1 (TCRGD) from T-cell Sciences (Cambridge, MA), Ber-H2 (CD30) from Dako (Glostrup, Denmark), and 1E9 (CD73; kindly provided by Dr M. Massaia,
University of Turin). For unconjugated MoAbs, an indirect immunofluorescence method was applied using an FITC affinity-purified
goat antimouse IgG antibody (Ortho).
For CD40 ligand (CD40L) and CD70 expression analysis, 2 X
IO5 CD3' or CD3-CD4' cells were cultured in 96-well flat-bottomed
microtiter plates (Nunc, Roskilde, Denmark) with PMA ( 5 ng/mL)
plus ionomycin (500 ng/mL) in a final volume of 200 & / W .
After activation (16 hours for CMOL and 96 hours for CD70
optimal expression), cells were harvested, washed two times with
phosphate-buffered saline (PBS)-5% FCS, and incubated for 30 minutes at 4"C, respectively, with 50 pL of biotin-conjugated antihuman
CD4OL-specific mouse MoAb (TRAP1; kindly provided by Dr R.
Kroczek, Robert Koch Institute, Berlin, Germany)2' or 50 pL of
biotin-conjugated antihuman CD70 (2F2; kindly donated by Dr
R.A.W. Van Lier, Central Laboratory of the Netherlands Red Cross
Blood Transfusion Service, Amsterdam, The Netherlands); cells
were then washed twice in PBS-FCS and incubated with PE-conjugated Streptavidin (Sigma).
After two washes in PBS-FCS, samples were analyzed with a
flow cytometer equipped with an argon-ion laser (488 nm; Cytoron
Absolute; Ortho) using the ABS software. Gates were selected on
lymphoid cells as determined by forward and right-angle scatter
properties; dead cells were excluded by setting an appropriate threshold trigger on the low forward light scatter parameter, and nonspecific staining was assessed using FITC-conjugated nonimmune
mouse IgG (Coulter). Data were acquired and stored in list mode.
Fluorescence analysis was performed on a log scale.
Cytokine assays. IL-4 and IFN-y were measured by sandwich
enzyme-linked immunosorbent assay (ELISA) as described.22The
mouse MoAb 8F1223and biotinylated MoAb 3H4 were used for IL4, MoAb 42-103 (kindly provided by Dr G. Delespesse, University
of Montreal, Montreal, Quebec, Canada) and biotinylated KM-48
(Kyowa Medex, Tokyo, Japan) were used for IFN-y determination.
The sensitivities of the IL-4 and the IFN-y ELISAs were 30 and 50
pg/mL, respectively. The binding to the second antibody was detected by stepwise incubation with alkaline phosphatase-conjugated
streptavidine (Tago 6567; Tago, Burlingame, CA) and 4-nitrophenyl
phosphate disodium salt (Merck, Darmstadt, Germany) and detected
at 405 nm in a Vmax kinetic microplate ,ELISA reader (Molecular
Devices, Sunnyvale, CA).
The IL-2 activity was measured by (,H)dThd uptake ofIL-2dependent CTLL cells.z4 Serial dilutions of rIL-2 (1 U biologic
activity = 0.1 ng/mL) or IL-2 containing supernatants were incubated with 3 X IO' CTLL cells in 200 pL for 24 hours, followed
by a 6-hour pulse of 1 pCi/well of ('H) dThd. This bioassay detects
50 pg/mL of human rIL-2.
IL-5 was estimated with a commercial ELISA-Kit (Quantikine;
R&D Systems, Minneapolis, MN) as recommended by the manufacturer.
Purification of eosinophils. Eosinophils were isolated from human blood by negative selection of granulocytes that do not express
CD16 incorporating a magnetic cell separation system (MACS; Miltenyi Biotec, Bergisch-Gladbach, Germany), as previously des ~ r i b e d . The
~ ~ . purity
~ ~ was 98% to 99%as determined by light
microscopy and flow cytometry. Cells were washed and resuspended
in complete culture medium to a density of 1 X 106/mL.
Eosinophil culture. Eosinophils were cultured in complete medium with 5% serum from the HES patient or from a control individual in the presence or absence of an antihuman IL-5 MoAb (IO pg/
mL; R&D Systems, Abingdon, UK) for 48 hours at 3 7 T , 5% COz
and 95% air in a humdified atmosphere.
Determination of eosinophil death by flow cytometry. The relative amounts of viable cells were determined by uptake of 1 mmoY
L ethidium bromide (Becton Dickinson, Mountain View, CA) in
dead cells by flow cytometric analysis."
DNA analysis. High molecular weight DNA extraction and purification from blood lymphocytes was performed as previously described in detail." DNA wasdigested with three restriction enzymes:
EcoRI, HindIII, and BamHI. Restriction fragments were electrophoretically size-separated on gel, transferred to nylon membrane that
was prehybridized, and hybridized with '*P-radiolabeledDNA
probes. The DNA probe used in T study was genomic probe of the
cDNA of the human TCRB chain second constant region gene (CTB,
0.5-kb fragment). Each gel included one lane containing size markers
composed of a HindIII digestion of X phage and a lane containing
high molecular weight human placenta DNA digested with the same
restriction enzymes corresponding to that ofthe other samples in
the gel. Hybridization and washings were performed under stringent
conditions, and autoradiographic exposures on Kodak X-AR x-ray
film (Eastman Kodak, Rochester, NY) were performed for 1 day.
RNA extraction and cDNA synthesis and amplification by poly-
From by guest on December 22, 2014. For personal use only.
merase chain reaction (PCR). Total RNAwaspreparedfrom
Tabb 1. Lymphocyte ImmunophonotypialAnalyrlr
CD3'CD4+ and CD3-CD4' T-cell subsets by the guanidiniumthioPatient PBL
cyanate-phenol-chloroformmethod.Onemicrogram ofRNAwas
Surface Markers
1% positive)
Normal Values
retrotranscribed in the first strand of the B-chain-specific compleA. Single-color studies
mentary DNA (cDNA) using the RiboClone cDNA
Synthesis System
Kit (Promega Corp, Madison,
W)and a primer specific
for TCRBCl
CTG CGG 3'). A
fraction of cDNA was subjected to enzymatic
amplification using a second human TCRBC primer (PA1 5' CCC
3') andaTCRBVdegenerated
primer [VPd 5' T(GIT)T(A/C~~)(C~~)T'GGTA(OT)(A/C)(A/G)(A/
T)CA 3'1 that was designed to amplify B-chain rearrangementsconTCRAB
taining virtually all the known human TCRBV genes?* The condiTCRGD
tions generate products of about 430 bp. PCR conditions were perCD16
formed under the following conditions: denaturation for 39 cycles
at 93В°C for 1 minute, annealing at 52В°C for 1 minute, and extention
CD1 1b
at 72В°C for 1 minute; the last cycle extention was performed at 72В°C
for 7 minutes. The specificity of the total amplified product was
specific probes.29
To preventsamplecontaminations,severalprecautionswere
taken. All solutions were aliqutated and used onlyonce and a negative control (PCR master mix with primersfor TCRBC and TCRBV
B. Two-color studies
degenerated without cDNAtemplate) was processed with TCR samples in each step of the experiments.In addition, eachT-cell sample
was amplified for the @actin gene to assess the integrity of the
mRNA extraction.
Lymphocytesu$acemarkeranalysis. Immunophenotypical studies showed the presence of a CD3-TCR-CD4+
population (Table 1A). All CD4+ cells wereCD2+CD5'
CD28+CD57-CDllb-CD25DRTD73- (Table 1B). Moreover, all CD4+ lymphocytes
present intheCD3-depleted
population (ie,CD3-CD4'
cells) were CD45RO+ (datanotshown).Unstimulated
CD3-CD4+ did not express CD40 ligand or CD30, whereas
CD70 was weakly expressed. After stimulation with PMA
plus ionomycin, normal expression of CD4OL and unusual
higher expression of CD70 compared with CD4+ cells of
eight normal controls (85% v 12.1% 2 6.6%) was found on
these cells (Fig 1).
Proliferative response. Proliferative response of total
patient PBMC after activation withPHAand PWM was
similar to that of healthy control, whereas it was reduced
after activation with CD3 and CD2 (Table 2). On the contrary, CD3-CD4+ enriched PBMC were not able to respond
to mitogen activating the cells through cross-linking of membrane receptors (notably PHA, CD3, and CD2), but proliferative response and expression of activation markers such as
CD69 and CD25 was partially restored when the transmembrane protein kinase C (PKC) activator PMA was added to
PHA or was used in combination with ionomycin.
I n vitro cytokine secretion. UnstimulatedPBMCand
PBMC depleted ofCD3' cells secrete very low levels of
IL-4, L-5, IL-2, and IFN-y, similar to the healthy control
(data not shown).
As shown in Fig 2, unfractionated PBMC from the patient
and healthy control produced similar levels of L-4, L-2,
and IFN-y after stimulation with PHA plus PMA. Removal
of CD3' cells from patients' PBMC with anti-CD3 MoAb
CD4'CDll 'b
Lymphocyte immunophenotypical analysis.
precoated magnetic beads abrogated the ability to produce
L - 2 and IFN-y,but did not modify significantly the levels
of L - 4 production. Of note, PBMC from patients produced
exceedingly high levels of L - 5 on stimulation with PMA
plus PHA compared with the healthy control. Also,this production was only partially reduced after removal of CD3+
cells, thus suggesting that this was essentially performed by
CD3-CD4+ cells.
Serum eosinophil survival factor study.Abioassay
eosinophil in vitro survival showed that patient's serum contained much higher amounts of eosinophil survival factors
than that of a healthycontrol, indicating that the CD3-CD4'
cells indeed produced cytokines with antiapoptotic properties for eosinophils. A neutralizing antihuman L-5 MoAb
partially blocked the effect of the patient's serum (Fig 3).
TCR analysis. Southern blot analysis of the DNA extracted from the patient's PBMC showed agermline configuration of the TCRB chain gene with no rearranged clonal
bands present on EcoRI-, BamHI-, or HindIII-digested preparations, as in the normal control (Fig 4). TCRBV repertoire
analysis performed by reverse transcription-polymerase
chain reaction of mRNA extracted from both CD3'and
CD3-CD4+ purified T-cell populations showed a polyclonal
From by guest on December 22, 2014. For personal use only.
Table 2. Proliferation Studies
(1.2 mglmL)
CD3 (60nglmL)
PWM (0.6 mglmL)
PMA (5 ng/mL)
c ionomycin
(500 nglmL)
PHA (1.2 mglmL) +
PMA (5 nglmL)
Healthy Control
770 2 9
2 95
72,850 c 1,050
c 35
13,300 -c 270
700 -c 58,450
2 680
2 7
20,700 c 920
-c 120
c 3,200
c 920
c 920
2 1,050
5,400 ND
2 150
Proliferation studies of patients' total and CD3-CD4' enriched
PBMC. Data are expressed as the mean cpm 2 SEM of a triplicate
Abbreviation: ND, not determined.
Fig 1. Expression of CD40L and CD70 on purified CD3-CD4' lymconphocytes of patient(left)and CD3TD4' lymphocytes of normal
trol (right). CD4' cells were obtainedby positiveselection with magnetic beads preceded by negative
with CD3-coated
magnetic beads as far as for the patients'
cells. The cells were resting
or activated with PMA (5 ng/mL) + ionomycin (500 ng/mL). Dashed
lines represent the fluorescence of cells stained with nonimmune
mouse IgG. Data are presented as relative log fluorescence intensity.
that observed is quite frequent in peripheral T-cell
lymphoma, being detected in 10% to IS% of
our study, MoAbs directed against two different epitopes of
the CD3 molecule were used to exclude the possibility that
lack ofrecognition of the CD3 antigen was caused by genetic
polymorphism; moreover, the lack of the CD3 molecule is
concordant with the lack of reaction with MoAbs directed
against the TCRAB and TCRGD frameworks.
A further remarkable phenotypic feature of this
CD3TD4' lymphocyte population was the expression (constitutively withlow density, strongly positively modulated
after stimulation withPMA plus ionomycin) of CD70, a
molecule involved in the regulation of T-cell proliferation
and survivaL3' CD70 isnot expressed on normal resting
CD4' cells and only marginally after stimulation (personal
TCRBV rearrangement in CD3' cells and no RNAtranscription in the CDTCD4' population (Fig 4); in this latter case,
the detection of,&actin amplification product showed the
integrity of the mRNA extraction and amplification procedure (data not shown).
We present a case of HES associated with the presence
of a cell population with an unusual CD3-, TCR-, CD4'
immunophenotype, without detectable TCRBV mRNA and
clonal rearrangement of TCRB gene, which was able to produce large amounts of IL-S after stimulation.
This cell population was made of mature T lymphocytes;
it had normal lymphocyte morphology and expressed T-cell
characteristic antigens (CD2, CDS, CD7, CD28, CD4SR0,
and, after in vitro stimulation, CD40L) but not markers (TdT,
CDla) of immature T cells. However, these cells lacked
surface CD3 and TCR expression. A phenotype similar to
Fig 2. Cytokine production by total
PBMC from (0)
healthy control
and from (m) totaland
(a) CD3-depleted (ie, enriched in the
CD3-CD4+ population) PBMC from patient in response t o PHA (1.2
pg/mL) PMA (5 ng/mL).
From by guest on December 22, 2014. For personal use only.
Despite its phenotypic abnormalities, therewasno evidence of clonality by Southern analysis of TCRB gene rearrangement. This was performed using three different restriction enzymes, making it unlikely that rearranged bands were
missed because of comigration of rearranged DNA fragments.32The detection of germline bands can be explained
by the presence of normal non-T cells in the population
studied (whole PBMC preparation).
Deletion of TCRB gene in an abnormal (clonal?) population would better explain the lack of TCRB gene rearrangement detection as well as the lack of mRNA transcription
and of the surface expression of its protein product, which
were shown in the present case. Indeed, the absence of clonal
TCRB gene rearrangement has been already described in
several cases of CD3- peripheral T-cell lymphomas and deletion of the gene has been suggested as a possible explanat i ~ n On
. ~ the
~ other hand, it is unlikely that these lymphocytes were of reactive nature. In fact, a stimulus should be
hypothesized that is able to induce simultaneously in multiple T-cell clones a failure of TCRBV mRNA transcription
and therefore the absence of membrane expression of
TCRAB (and CD3). No normal population with these features has been described. We therefore believe that the abnormal lymphocyte population of this patient was the expression of a peripheral T-cell lymphoma with a very indolent
clinical course.
Interestingly, lymphocytes with the same unusual phenotype were described inpatients in whom the clinical onset of
T-cell lymphoma was preceded by a several-year history of
eosinophilia''.I3 and ina recently reportedcase of eosinophilia,
9 E 9
.- I5n .-
Fig 3. Effect of sera from the patient withHES or from a normal
control individual in the presence or absence of an a n t i - I l l MoAb
on thesurvival ofeosinophils. Eosinophils were culturedin complete
medium with 5% serum. Viable eosinophils were measured after
48 hours. Data represent mean values of one of three independent
experiments. Each experiment was performedin duplicate and also
in complete medium with 10% serum, which showed identical results.
1 2 3 4
24 Kb +
11 Kb +
10,sKb +
3,s Kb
Barn HI Eco RI
Hind Ill
Fig 4. (A) Southern blot analysis of gene coding for theB chain of theT-cell receptor. DNA from patient PBMC was digested with EcoRl
(lane 3). BamHl (lane 6). and Hindlll (lane 9) restriction enzymes and hybridized t o G Bprobe. Each gel included one lane containing size
markers composed of a Hindlll digestion of k phage and a lane containing high molecular weight human placenta DNA digested with the
same restriction enzymes corresponding t o that of the other samples in the gel. (B) RT-PCR amplification of mRNA from CD3' (lane 1) and
CD3-CD4' (lane 2) cells of patient. Lane 3 is the negativePCR control, whereas lane 4 contains synthetic DNA size marker pBR 322 digested
Methods, using the human
TCRBC primer (PA11and the TCRBV
with Hae 111. The amplification was performedas described in the Materials and
TCRBV genes. This
degenerate primer (Vpd) that wasdesigned t o amplify p chain rearrangements containing virtually all the known human
generates products of about 430 bp, as indicated by the arrow.
From by guest on December 22, 2014. For personal use only.
in which the abnormal lymphocytes had a typical Th2 cytokine secretion pattern, were clonal, but were not clinically
lymphomatous.2oThis case was described as the first example
of a proliferation of Th2 lymphocytes in humans.
Also in the present case, evaluation of in vitro cytokine
production suggested that the abnormal T lymphocytes were
responsible for the eosinophilia through the production of
IL-5. This was supported by the demonstration of eosinophil
survival factors in the patients’ serum, whose activity was
partially neutralized by an antihuman IL-5 antibody.
Their cytokine secretion pattern suggested that they belonged to the Th2 helper lymphocyte subgroup because they
did not secrete either IL-2 or IFN-7Unlike
the case reported
by Cogan et al,” our patient’s cells did not produce high
levels of IL-4, in accordance with the low serum IgE level
of the patient. This feature did not preclude the assignment
of these cells to the Th2 lymphocyte subgroup. Indeed, it
has been recognized that the pattern of cytokine secreted by
human T-cell clones may be more complex than that of a
pure ThlRh2 profile, and it seems that different environmental factors could be responsible for the functional polarization of T-cell clones towards a particular b e h a ~ i o rAlthough
common mechanisms in the control expression of IL-4 and
IL-5 have been shown, the possibility of a differential regulation has also been reported (eg, IL-2 induces IL-5 but not
IL-4 mRNA expression in mouse T cells).33In vivo, a dissociation between IL-4 and IL-5 secretion was reported in
nonatopic asthmatic patients.% The lack of TCWCD3 complex may further explain, in our study, this dissociation, as
suggested by the ability of cyclosporin to inhibit IL-4 but
not IL-5 CD3-induced prod~ction.’~
Another peculiar feature of this case was the absence of
spontaneous in vitro secretion of IL-5, suggesting that persistent cell activation byan
as yet unknown stimulus
was responsible for its in vivo production. Although the
CD3TD4+ lymphocytes were not able to respond to signals
acting through the TCFUCD3 complex, they expressed different structures (such as CD2 and CD28) related to the transduction of T-cell activating signals. Studies are in progress
to see if the abnormal expression of CD70 by these cells
could also play a role in this context.
In conclusion, we believe that this is a case of HES pathogenetically linked to the presence of an abnormal population
of Th2 helper cells, which was able to selectively secrete
large amounts of L - 5 . From a clinical standpoint we will
carefully observe the patient because a malignant disease
may appear several years after the recognition of eosinophilia.ll.’3-15Moreover, because in this case the absolute lymphocyte count was within normal range, we suggest that a
lymphocyte immunophenotypic analysis should be included
in the diagnostic workup of every case of HES to detect the
presence of even small subpopulations withan abnormal
phenotype. Future studies should determine whether there is
a specific association between eosinophilia and the
CD3-CD4+ phenotype of lymphocytes, as suggested by this
and several other reports.’1,13*20
1. Basten A, Beeson PB: Mechanism of eosinophilia. 11. Role of
the lymphocyte. J Exp Med 131:1288, 1970
2. Weller PF, Bubley GJ: The idiopathic hypereosinophilic syndrome. Blood 83:2759, 1994
3. Romagnani S: Human Thl and Th2 subsets: Doubt no more.
Immunol Today 12:256, 1991
4. Sanderson CJ: Interleukin-5, eosinophils and disease. Blood
79:3101, 1992
5. Romagnani S: Lymphokine production by human T cells in
disease states. AMU Rev Immunol 12:227, 1994
6. Owen W,
Rothenberg ME, Petersen J, Weller PF, Silberstein
D, Sheffer AL, Stevens RL, Soberman RJ, Austen KF: Interleukin
5 and phenotypically altered eosinophils in the blood of patients
with the idiopathic hypereosinophilic syndrome. J Exp Med 170:343,
7. Enokihara H, Kajitani H, Nagashima S, Tsunogake S, Takano
N, Saito K, Furusawa S, Shishido H, Hitoshi Y, Takatsu K: Interleukin 5 activity in sera from patients with eosinophilia. Br J Haematol
75:458, 1990
8. Screzenmeier H, Home SD, Tewald F, Fleischer B, Raghavachar A: Interleukin-5 is the predominant eosinophilopoietin produced
by cloned T lymphocytes in hypereosinophilic syndrome. Exp Hematol 21:358, 1993
9. Simon HU, Blaser K Inhibition of programmed eosinophil
death: A key pathogenic event for eosinophilia? Immunol Today
16:53, 1995
10. Yousefi S, Green Dr, Blaser K, Simon HU: Protein tyrosin
phosphorylation regulates apoptosis in human eosinophils and neutrophils. Proc Natl Acad Sci USA 91:10868, 1994
11. O’Shea JJ, Jaffe ES, Lane HC, MacDermott RP, Fauci AS:
Peripheral T-cell lymphoma presenting as hypereosinophilia with
vasculitis. Am J Med 82:539, 1987
12. Chott AC, Augustin I, Wrba F, Hanak H, Ohlinger W. Radaskiewicz T: Peripheral T-cell lymphomas: A clinico-pathological
study of 75 cases. Hum Pathol 21:1117, 1990
13. Bagot M, Bodemer C, Wechsler J, Divine M, Haioun C,
Capesius C, Saal F, Cabotin P, Roubertie E, De Prost Y, Lorette G,
Revuz J: Lymphome T non dpidermotrope prdctdd pendant plusieurs
anntes par un syndrome hyperkosinophilique. Ann Dermatol Venereol 117:883, 1990
14. Moraillon I, Bagot M, Bournerias I, Prin L, Wechsler 3, Revuz
J: Syndrome Hypereosinophilique avec pachydermie prhddant un
lymphome. Traitement par l’interfdron alpha. Ann Dermatol Venereol 118:883, 1991
15. Kim CJ, Park SH, Chi JG: Idiopathic hypereosinophilic syndrome terminating as disseminated T-cell lymphoma. Cancer
67:1064, 1991
16. Abruzzo LV, Jaffe ES, Coteligam JD, Whang-Peng J, DelDuca VJ, Medeiros LJ: T-cell lymphoblastic lymphoma with eosinophilia associated with subsequent myeloid malignancy. Am J Surg
Pathol 16:236, 1992
17. Murata K, Yamada Y, Kamihara S, Atogami S, Tsukasaki K,
Momita S, Amagasaki T, Sadamori N, Tomonaga M, Kinoshita KI,
Ichimaru M: Frequency of eosinophilia in adult T-cell leukemid
lymphoma. Cancer 69:966, 1992
18. Vowels BR, Cassin M, Vonderheid EC, Rook AH: Aberrant
cytokine production by Sezary syndrome patients: Cytokine secretion pattern resembles murine Th2 cells. J Invest Dermatol 99:90.
19. Samoszuk M, Ramzi E, Cooper DL: Interleukin-5 mRNA in
three T-cell lymphomas with eosinophilia. Am J Hematol 42:402,
20. Cogan E, Schandend L, Crusiaux A, Cochaux P,Velu T,
Goldman M: Clonal proliferation of type-2 helper T cells in a man
with the hypereosinophilic syndrome. N Engl J Med 330:535, 1994
21. Brugnoni D, Air6 P, Graf D, Marconi M, Lebowitz M, Plebani
A, Giliani S, Malacarne F, Cattaneo R. Ugazio AG, Albertini A,
From by guest on December 22, 2014. For personal use only.
Kroczek RA, Notarangelo LD: Ineffective expression of CD40 ligand on cord blood T cells may contribute to poor immunoglobulin
production in the newborn. Eur J Immunol241919, 1994
22. Carballido JM, Carballido-Perrig N. Terres G, Heusser CH,
Blaser K: Bee venom phospholipase A2-specific T cell clones from
human allergic and non-allergic individuals: Cytokine patterns
change in response to the antigen concentration. Eur J Immunol
22:1357, 1992
23. Andersson U, Andersson J, Lindfors A, Wagner K, Moller G,
Heusser CH: Simultaneous production of interleukin 2, interleukin 4
and interferon-? by activated human blood lymphocytes. Eur J Immunol 20:1591, 1990
24. Gillis S, Ferm MM, Du W, Smith KA: T cell growth factor:
Parameters of production and a quantitative microassay for activity.
J Immunol 120:2027, 1978
25. Simon HU, Tsao PW, Siminovitch KA, Mills GB, Blaser
K Functional platelet-activating factor receptors are expressed by
monocytes are expressed by monocytes and granulocytes but not by
resting or activated T and B lymphocytes from normal individuals
or patients with asthma. J Immunol 153:364, 1994
26. Yousefi S, Hemman S, Weber M, Hoelzer C, Hartung K,
Blaser K, Simon HU: IL-8 is expressed by human peripheral blood
eosinophils. Evidence for increase secretion in asthma. J Immunol
154:5481, 1995
27. Flug F, Pelicci PG, B o n d F, Knowles DMI, Dalla Favera R: T
cell receptor gene rearrangement as markers of lineage and clonality in
T cell neoplasms. P m Natl Acad Sci USA 823460,1985
28. Martin R, Howell MD, Jaraquemada D, Flerlage M, Richert
J, Brostoff S, Long EO, McFarlin DE, McFarland HF: A myelin
basic protein peptide is recognized by cytotoxic T cells in the context
of four HLA-DR types associated with multiple sclerosis. J Exp
Med173:19, 1991
29. Bettinardi A, Imberti L, Sottini A, Primi D: Analysis of amplified T cell receptor VD transcripts by a non-isotopic immunoassay.
J Immunol Methods 146:71, 1992
30. Picker LJ, Brenner MB, Weiss LM, Smith SD, Wamke RA:
Discordant expression of CD3 and T-cell receptor beta-chain antigens in T-lineage lymphomas. Am J Pathol 129:434, 1987
31. Hintzen RQ, de Jong R, Lens SMA, van Lier RAW: CD27:
Marker and mediator of T-cell activation? Immunol Today 15:307,
32. Weiss LM, Picker W ,Grogan TM, Wamke RA, Sklar J:
Absence of clonal beta and gamma T-cell receptor gene rearrangements ina subset of peripheral T-cell lymphomas. Am J Pathol
130:436, 1988
33. Bohjanen PR, Okaijma M, Hodes RJ: Differential regulation
of interleukin-4 and interleukin 5 gene expression: A comparison of
T-cell induction by anti-CD3 antibody or by exogenous lymphokines. Proc Natl Acad Sci USA 875283, 1990
34. Walker C, Bode E, Boer L, Hansel TT, Blaser K, Virchow
JC Jr: Allergic and nonallergic asthmatics have distinct patterns of
T-cell activation and cytokine production in peripheral blood and
bronchoalveolar lavage. Am Rev Respir Dis 146:109, 1992
From by guest on December 22, 2014. For personal use only.
1996 87: 1416-1422
A case of hypereosinophilic syndrome is associated with the
expansion of a CD3-CD4+ T-cell population able to secrete large
amounts of interleukin-5
D Brugnoni, P Airo, G Rossi, A Bettinardi, HU Simon, L Garza, C Tosoni, R Cattaneo, K Blaser
and A Tucci
Updated information and services can be found at:
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
Information about ordering reprints may be found online at:
Information about subscriptions and ASH membership may be found online at:
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.
Без категории
File Size
1 705 Кб
Пожаловаться на содержимое документа