Intercellular adhesion molecule-1 is upregulated on peripheral blood T lymphocyte subsets in dual asthmatic responders.

Abstract

To examine the role of adhesion molecules in T cell recruitment and activation during allergen-induced late asthmatic response (LAR), we evaluated the expression of lymphocyte function-associated antigen-1 alpha (LFA-1 alpha) and intercellular adhesion molecule-1 (ICAM-1) on peripheral blood T lymphocyte subsets from atopic asthmatic patients and their changes following allergen inhalation challenge. 12 atopic asthmatic patients were studied. Six patients showed only a single early response after allergen challenge, and six developed a dual response. At baseline, dual responders (DR) had a significantly higher expression of ICAM-1 on CD4+ and CD8+ T lymphocytes as compared with both single early responders (P < 0.005 and P < 0.02, respectively) and controls (P < 0.001, both comparisons). Allergen challenge was followed by a decrease of CD8+ ICAM-1+ T lymphocytes in all DR (P < 0.05) and of CD4+ ICAM-1+ T lymphocytes in four out of six DR, at the time of the LAR. At the same time, a significant rise in serum levels of the soluble form of ICAM-1 was observed in DR. These results suggest that peripheral blood immunoregulatory T lymphocytes are in a higher state of activation in DR as compared with early responders. The upregulation of ICAM-1 on these cells may be important in enhancing airway inflammation in patients with LAR.

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Intercellular Adhesion Molecule-I Is Upregulated on Peripheral Blood T
Lymphocyte Subsets in Dual Asthmatic Responders
Virginia De Rose,* Giovanni Rolla,* Caterina Bucca,* Paolo Ghio,* Massimo Bertoletti,* Paolo Badema,*
and Emesto Pozzi*
*Department of Clinical and Biological Sciences, Respiratory Disease Division, and tDepartment ofBiomedical Sciences and Human
Oncology, University of Turin, Turin 10100, Italy
Abstract
To examine the role of adhesion molecules in T cell recruit-
ment and activation during allergen-induced late asthmatic
response (LAR), we evaluated the expression of lymphocyte
function-associated antigen-la (LFA-1a) and intercellular
adhesion molecule-i (ICAM-1) on peripheral blood T lym-
phocyte subsets from atopic asthmatic patients and their
changes following allergen inhalation challenge. 12 atopic
asthmatic patients were studied. Six patients showed only a
single early response after allergen challenge, and six devel-
oped a dual response. At baseline, dual responders (DR)
had a significantly higher expression of ICAM-1 on CD4'
and CD8' T lymphocytes as compared with both single early
responders (P < 0.005 and P < 0.02, respectively) and con-
trols (P < 0.001, both comparisons). Allergen challenge was
followed by a decrease of CD8' ICAM-1l T lymphocytes in
all DR (P < 0.05) and of CD4' ICAM-1l T lymphocytes in
four out of six DR, at the time of the LAR. At the same
time, a significant rise in serum levels of the soluble form
of ICAM-1 was observed in DR. These results suggest that
peripheral blood immunoregulatory T lymphocytes are in
a higher state of activation in DR as compared with early
responders. The upregulation ofICAM-1 on these cells may
be important in enhancing airway inflammation in patients
with LAR. (J. Clin. Invest. 1994.94:1840-1845.) Key words:
adhesion molecules * lymphocytes * airway inflammationX
asthma * allergen.
Introduction
There is increasing evidence that T lymphocytes play a central
role in immune-inflammatory response in asthma. These cells
recognize and respond directly to allergens and, through the
release of several cytokines, may orchestrate the inflammatory
response in asthma (1). T lymphocyte activation has been dem-
onstrated in peripheral blood in both stable and acute severe
asthma (2-5) and in bronchoalveolar lavage of patients with
mild to moderate disease severity (5-7). Activated T cells have
Address correspondence to Virginia De Rose, M.D., Clinica di Malattie
dell'Apparato Respiratorio, Dipartimento di Scienze Cliniche e Biolo-
giche, Universith di Torino, Ospedale S. Luigi Gonzaga, Regione Gon-
zole 10, 10043 Orbassano (Torino), Italy.
Receivedfor publication 12 April 1994.
also been identified in the bronchial mucosa of atopic asthmat-
ics (8).
A critical initial step in inflammatory cell migration from
vascular compartments into the airways is the expression of cell
surface adhesion molecules. A large number of these molecules
have been identified and characterized in recent years (9-12).
Among T lymphocyte adhesion receptors, the heterodimer
lymphocyte function-associated antigen-I (LFA-1)' and the in-
tercellular adhesion molecule-i (ICAM-1) have been shown to
play an important role not only in T cell recruitment, but also
in T cell activation and in the development of specific immune
responses (10, 13-16). LFA-1 is a member of the 62 family of
integrins, which is constitutively expressed on most leukocytes
(9-14). ICAM-1 is a cell adhesion molecule, belonging to the
immunoglobulin supergene family, which is expressed on a
variety of hemopoietic and nonhemopoietic cells, and is upregu-
lated at sites of inflammation (10, 17, 18). ICAM-1 is expressed
only weakly on resting T lymphocytes, but activation of these
cells by mitogens or specific cytokines increases its expression
(15, 17-19).
Recent studies suggest that the interaction between ICAM-
1 and LFA-l plays a role in the development of allergic airway
inflammation (20-24).
The allergen-induced late asthmatic response (LAR) may
provide a useful model for investigating inflammatory events
in atopic asthma. The LAR is associated with airway inflamma-
tion and it has been suggested that the recruitment and activation
of inflammatory cells lead to local changes in the airways (25-
28). Recent studies have demonstrated changes in T cell subsets
in both peripheral blood and bronchoalveolar lavage after aller-
gen challenge, suggesting a link between T lymphocyte function
and the occurrence of the LAR (29-31).
We speculated that enhanced expression of adhesion mole-
cules on circulating T cells might be relevant to the selective
recruitment and activation of these cells.
To test this hypothesis we evaluated the expression of LFA-
la and ICAM-1 on peripheral blood T cell subsets from atopic
asthmatic patients with or without LAR and their changes fol-
lowing allergen inhalation challenge. Normal volunteers were
evaluated as controls for baseline data. Moreover, as a soluble
form ofICAM-1 has recently been described (32, 33) and shown
to increase in acute asthma and other inflammatory conditions
(34, 35), we also determined the serum levels of the soluble
form of ICAM-1 (s-ICAM-1) in asthmatic patients and control
subjects.
1. Abbreviations used in this paper: DR, dual responder; ER, early
single responder; FEVy, forced expiratory volume in 1
intercellular adhesion molecule-i; LAR, late asthmatic response; LFA-
1, lymphocyte function-associated antigen-i; PE, phycoerythrin; s-
ICAM-1, soluble ICAM-1.
s; ICAM-1,
1840
V. De Rose, G. Rolla, C. Bucca, P. Ghio, M. Bertoletti, P. Baderna, and E. Pozzi
J. Clin. Invest.
C The American Society for Clinical Investigation, Inc.
0021-9738/94/11/1840/06
Volume 94, November 1994, 1840-1845
$2.00

Page 2

Methods
Patients.
women), 16 to 34 yr of age, were studied. The patients were selected
on the basis of (a) positive skin prick tests to common inhalant allergens
(Neo Abello, Ospiate di Bollate, Italy) and increased specific IgE for
the relevant allergen; (b) baseline forced expiratory volume in 1 s (FEVI)
> 81% of predicted value (36); (c) increased bronchial responsiveness
to inhaled methacholine (i.e., provocative dose required to decrease the
FEVy by 20% of its baseline value [PD20 methacholine] below 800 jig);
(d) stable clinical conditions (i.e., no acute asthmatic attack and no
respiratory tract infection in the last 2 mo); (e) no treatment other than
rescue /32-adrenergic drugs, which were discontinued at least 24 h before
the study. All subjects were informed on the nature and the scope of
the study and gave written consent.
10 normal volunteers (6 men and 4 women), 24 to 40 yr of age,
were evaluated as controls for baseline data. Normal volunteers were
asymptomatic, had negative skin prick tests, normal total IgE levels, no
detectable serum allergen specific IgE to common aeroallergens, and had
normal responsiveness to methacholine. All subjects were nonsmokers.
Allergen inhalation challenge. Spirometry was recorded using a
computerized water-sealed spirometer (BAIRES; Biomedin, Padova, It-
aly). Forced vital capacity and FEV, were calculated from the best
curve. The allergen inhalation challenge was performed following the
guidelines of the American Academy of Allergy (37). For each patient,
the allergen producing a positive immediate skin response was chosen
for the challenge. Eight patients received grass pollen and four patients
received Dermatophagoides pteronyssinus. Allergen solutions were
freshly prepared using lyophilized allergens (Neo Abellb) diluted in
sterile PBS.
The solutions were delivered by a compressed air nebulizer, con-
trolled by a breath-actuated dosimeter (MEFAR MB3; Markos, Monza,
Italy). The dosimeter was set to nebulize for
1 s of nebulization).
After recording baseline spirometry, patients inhaled from the nebu-
lizer five breaths of PBS from functional residual capacity to total lung
capacity. FEVY
was recorded 5 min after the inhalation. Thereafter,
they inhaled an initial dilution of 1:10,000 allergen solution (titrated in
biological units). FEVI was recorded 10 min after each set of inhalations.
IfFEV, had not fallen by 20% from post-PBS value, allergen inhalation
was continued with incremental 10-fold concentrations, until a FEV,
decrease of at least 20% of the post-PBS value was obtained. The
provocation dose required to reduce FEV, by 20% of the post-PBS
value (PD20 allergen) was derived by linear interpolation. FEV, was
measured at 5, 10, 15, 30, 45, and 60 min after challenge, and then
hourly for as long as 10 h. Patients were designated as either single early
responders (ER) or dual (early and late) responders (DR) depending on
whether they developed a 15% or greater decrease in FEV, during the
3-10 h period after the initial early-phase reaction. After 10 h the
patients returned home. They were instructed on how to record hourly
their peak expiratory flow rate using a portable peak flow meter and
what to do in case of respiratory complaints.
Isolation oflymphocytes. Peripheral venous blood was obtained from
each subject immediately before allergen challenge (baseline), and at
15 min and 6 h after challenge.
Total white blood cell count was determined with an automated cell
counter and the differential cell count was determined by examination
of Wright-stained slides.
Peripheral blood mononuclear cells were isolated from heparinized
venous blood by a standard Ficoll-Hypaque (Pharmacia, Uppsala, Swe-
den) density gradient technique. The mononuclear cell-rich fraction was
collected from the plasma/Ficoll interface and washed twice with RPMI
1640 (GIBCO, Paisley, Scotland). Lymphocytes were then obtained
by removal of plastic-adherent cells. Briefly, mononuclear cells were
incubated for 1 h at 37°C on plastic petri dishes (Nunc, Roskilde, Den-
mark); nonadherent cells were then removed, washed again with RPMI
1640 and resuspended in PBS containing 0.1% sodium azide and 0.5%
BSA (PBS/BSA) for marker analysis.
12 nonsmoking atopic asthmatic patients (7 men and 5
1 s (output 10±0.6ILper
Table I. Characteristics of the Asthmatic Patients
Parameter
Single responders
Dual responders
Number
Age (yr)
Sex (M/F)
Antigen
6
6
26.1±2.81
4/2
4: Grass pollen
2: D. pteronyssinus
28.3±1.59
3/3
4: Grass pollen
2: D. pteronyssinus
Baseline FEV1
(% predicted)
Early FEVI (% baseline)
Late FEV1 (% baseline)
PD20 methacholine (jg)
P20 allergen
(biological units)
102.3±5.62
78.72±1
91.4±1.79
171.6±119.7
106.6±1.59
65.9±7.5
74.9±4.1
147.7±112.7
0.48±0.18
0.31±0.11
Data are expressed as mean±SEM.
Flow cytometry. Adhesion molecule expression on T lymphocyte
subsets was determined by flow cytometric analysis after dual labeling
with the following murine IgG monoclonal antibodies: anti-CD4 or anti-
CD8 coupled to phycoerythrin (PE) (Becton-Dickinson, Cowley, UK),
anti-LFA-1a (CD1 la) or anti-ICAM-1 (CD54) coupled to FITC (Immu-
notech, Marseille, France). Negative controls consisted of isotype-
matched antibodies with irrelevant specificities (Becton-Dickinson) and
were used in all experiments.
Aliquots of 500,000 cells in 50 jl of PBS/BSA were incubated for
30 min with 10Alof one of each of the FITC-labeled antibodies along
with 10 jl of anti-CD4-PE or anti-CD8-PE. After washing once in
PBS/BSA, the cells were resuspended in 1% paraformaldehyde for anal-
ysis. All steps were performed at 40C.
Flow cytometry was performed on a FACS Analyzer (Becton-Dick-
inson). Results were expressed as percent positive cells relative to an
isotype control antibody. One aliquot of cells was stained with CD4-
PE or CD8-PE and CD3-FITC (Becton-Dickinson) to confirm that all
CD4 or CD8 cells analyzed were T lymphocytes.
s-ICAM-J assay. Serum samples from asthmatic patients and control
subjects were analyzed for s-ICAM-1 using an ELISA. s-ICAM-1
ELISA kits were purchased from Bender MedSystems (Vienna, Austria).
The concentrations of s-ICAM-1 in serum were assayed according to the
procedure recommended by the manufacturer. Results were expressed as
ng/ml, relative to a s-ICAM-1 standard.
Statistical analysis. Statistical analysis was performed using nonpar-
ametric tests: Mann-Whitney U test for nonpaired data, to compare
baseline data in the two groups of asthmatic patients and control sub-
jects; and Wilcoxon's matched pairs test for within-group comparisons.
Correlations were determined by the Spearman's rank correlation test.
Data are expressed as mean±SEM.
Results
Characteristics of the asthmatic patients
The clinical characteristics of the asthmatic patients are shown
in Table I. A total of six patients showed only a single early
asthmatic response after allergen challenge, and six patients
developed a dual asthmatic response, within 6 h from allergen
inhalation.
The specific antigen to which the subjects were sensitized
was grass pollen, in most of the patients. Only two subjects
with single early response and two subjects with dual response
were sensitized to D. pteronyssinus. Age, baseline lung func-
tion, PD20 methacholine, and PD20 allergenwere similar in the
Intercellular Adhesion Molecule-i in Dual Asthmatic Responders
1841

Page 3

Table II. Absolute Counts and Percentages of CD4' and CD8' T
Lymphocytes and CD4/CD8 Ratio in Asthmatic Patients and
Control Subjects
Markers
Group
CD4*
CD8*
CD4/CD8
Single responders
Baseline
0.94±0.14
(45.5±2.89)
0.46±0.09
(30.6±1.91)
1.53±0.14
After challenge
15 min
0.92±0.14
(48.6±2.27)
1.07±0.20
(48.7±2.02)
0.51±0.07
(31±2.23)
0.56±0.04
(31.2±1.75)
1.61±0.20
6 h
1.60±0.15
Dual responders
Baseline
0.78±0.08
(47.9±4.1)
0.56±0.06
(30.6±2.5)
1.64±0.23
After challenge
15 min
0.83±0.06
(49.9±3.58)
0.95±0.14
(48.5±3.77)
0.52±0.07
(31.7±2.2)
0.64±0.10
(29.2±1.1)
1.63±0.22
6 h
1.68±0.10
Controls
Baseline
0.82±0.11
(47.3±1.97)
0.40±0.08
(29.9±1.2)
1.61±0.12
*Absolute counts (x 0-9/Iliter) and percentages (in parentheses) of pe-
ripheral blood CD4' and CD8' T lymphocytes in single responders (n
= 6), dual responders (n = 6), and controls (n = 10). Data are expressed
as mean±SEM.
two groups. Although the early maximum FEVy fall was larger
in DR than in ER, the difference was not statistically significant.
Flow cytometry
Baseline data. The absolute numbers and the percentages ofCD4'
and CD8' T lymphocytes as well as the CD4/CD8 ratio did not
differ in ER, DR, and control subjects at baseline (Table II).
The analysis ofadhesion molecule expression onT lymphocyte
subsets showed that dual asthmatic responders had a significantly
higher percentage of CD4' T lymphocytes expressing ICAM-1
when compared with single early responders (P < 0.005) and
control subjects (P < 0.001); in contrast, no significant differences
were observed between the latter two groups (Fig. 1 A). Even if to
a lesser extent, the percentage ofCD8' T lymphocytes expressing
ICAM-1 was also significantly higher in dual asthmatic responders,
compared with single responders (P < 0.02) and control subjects
(P < 0.001). No significant differences were observed between
ER and control subjects (Fig. 1 B).
Baseline values of both CD4' ICAM-lI
1 + subpopulations from the overall asthmatic patients were sig-
nificantly related to the changes in FEV, at the time of the LAR
(r=0.636, P < 0.03 and r= 0.629, P < 0.03, respectively),
whereas no correlations were observed with baseline FEVI,
PD20 methacholine, or PD20 allergen.
As shown in Table Ill, no differences were observed in the
percentage of CD4+ LFA-la+ or CD8+ LFA-la+ T lympho-
cytes between single early and dual asthmatic responders and
between the two groups of patients and control subjects, at
baseline (P > 0.05, all comparisons).
and CD8' ICAM-
50
45
40 -
35
-
-
co
-J
-Jw
C.)
'
i
6
30
25
20 -
15
10 -
5-
0 -
-
o
0
8
50
45
40
35
30 -
25
20
15
10
5
0 -
-
-
cn
-jw
-
-
+-
i
-
C
o
-
-
8
-
-
A
_0.005 I_I
p O.001
0
6
0
SINGLE
RESPONDERS
DUAL
RESPONDERS
k
CONTROLS
B
,
p o.o2
,
p o.oO1
BI------I~~
0
0
0
0
SINGLE
RESPONDERS
DUAL
RESPONDERS
0
CONTROLS
Figure 1. Baseline percentages of peripheral blood CD4+ ICAM-1+ T
lymphocytes (A) and CD8+ ICAM-1+ T lymphocytes (B) in single asth-
matic responders (n=6), dual asthmatic responders (n=6), and control
subjects (n = 10). Bars represent mean values.
Data after challenge. After allergen challenge, the percent-
age of CD8+ T lymphocytes expressing ICAM-1 significantly
decreased in dual asthmatic responders, at 6 h, compared with
baseline values and values at 15 min (P < 0.05 for both compar-
isons) (Fig. 2). However, no correlation was found between
changes of CD8+ ICAM-l T lymphocytes and FEV1 changes
at the time of the LAR.
There was a remarkable decrease in the percentage ofCD4+
ICAM-I' T lymphocytes in four out of six dual responders at 6
h after challenge. However, the decrease did not reach statistical
significance in the overall population of DR. No significant
changes in the percentage ofCD4+ ICAM-1+ and CD8+ ICAM-
1+ T lymphocytes were observed after challenge in patients
with single early response (Fig. 2).
The absolute numbers and the percentages of CD4+ and
CD8+ T lymphocytes as well as the CD4/CD8 ratio did not
change, after allergen challenge, either in ER or DR (Table II).
Similarly, no changes were observed in the percentage ofCD4+
or CD8+ T lymphocytes expressing LFA-la (Table III).
s-ICAM-J assay
Baseline serum levels of s-ICAM-l were not significantly dif-
ferent between the two groups of asthmatic patients. Similarly,
1842
V. De Rose, G. Rolla, C. Bucca, P. Ghio, M. Bertoletti, P. Baderna, and E. Pozzi

Page 4

Table III. Percentages ofCD4' LFA-Ja' and CD8' LFA-la' T
Lymphocytes in Asthmatic Patients and Control Subjects
Markers
Group
CD4' LFA-1a'
CD8' LFA-1a'
Single responders (n = 6)
Baseline
After challenge
15 min
6 h
Dual responders (n = 6)
Baseline
After challenge
15 min
6 h
Controls (n = 10)
Baseline
42.6±3.64
28.97±1.90
47.03±1.94
47.8±1.50
29.2±2.70
29.1±1.23
47.5±4.00
28.2±2.19
47.1±3.13
48.2±2.06
29.7±2.39
27.7±1.77
43.9±2.75
28±0.90
Data are expressed as mean±SEM.
no differences were observed between either ER and DR and
control subjects (ER: 288.68±28.49 ng/ml, DR: 281.23±12.88
ng/ml, C: 322.37±21.21 ng/ml; P > 0.05, all comparisons).
After allergen challenge, serum levels of s-ICAM-I signifi-
cantly increased in dual asthmatic responders, at 6 h, compared
with baseline values and values at 15 min (P < 0.03 for both
comparisons) (Fig. 3).
Discussion
This study was designed to evaluate the expression of the adhe-
sion molecules LFA-la and ICAM-1 on peripheral blood T
lymphocyte subsets from atopic asthmatic patients and their
changes after allergen inhalation challenge. The main findings
are: (a) dual asthmatic responders, at baseline, had an increased
expression ofICAM-1 on CD4' T lymphocytes and, to a lesser
extent, on CD8' T lymphocytes as compared with both single
early responders and control subjects, and (b) allergen challenge
was followed by a marked decrease of CD8' ICAM-1+ T lym-
phocytes in all DR and of CD4' ICAM-1+ T lymphocytes in
four out of six DR at the time of the LAR. At the same time,
a significant rise in serum levels of the soluble form of ICAM-
1 occurred in dual asthmatic responders.
ICAM-1 is a cell adhesion molecule expressed on a variety
of hemopoietic and nonhemopoietic cells, and upregulated by
inflammatory mediators (10, 15, 17-19). It is the counterrecep-
tor for the leukocyte integrins LFA-1 and Mac-i (38-41). The
relevance of ICAM-1/LFA-1 interaction in the development of
allergic airway inflammation is provided by the observation that
anti-ICAM-1 monoclonal antibodies are able to inhibit both
inflammatory cell infiltrate and acquired bronchial hyperrespon-
siveness accompanying repeated allergen challenge in nonhu-
man primate models (20). Furthermore, in nasal biopsies from
patients with perennial allergic rhinitis, ICAM-1 expression on
endothelial cells was shown to be significantly increased, com-
pared with healthy controls (23). Upregulation of ICAM-1 has
also been observed in human skin 6 h after local intradermal
challenge, in association with tissue eosinophilia (42).
Recent studies have shown that ICAM-1ILFA-1 interaction
SINGLE RESPONDERS
50-
40-
-Jw
° 30-
o 20-
o
0-
50-
40-
Co
-Jwo 30-
I--
Q 20-
8:
0
2 10-
0
15mh
After challenge
6h
Baseline
SINGLE RESPONDERS
50-
40-
w
' 30-
+
. 20-
0
at 10
0-
50-
40-j
CI
wo 30-
i
9 20-
-
0
0-
15min
After challenge
6h
Baseline
DUAL RESPONDERS
15min
Afterchallenge
6h
Baseline
DUAL RESPONDERS
15min
Afterchallenge
6h
Baseline
Figure 2. Changes in peripheral blood CD4' ICAM-I+ T lymphocytes
(top panels) and CD8' ICAM- + T lymphocytes (bottom panels) in
single asthmatic responders (n = 6) and dual asthmatic responders (n
= 6) after allergen inhalation challenge. The percentage ofCD8' ICAM-
1 + T lymphocytes significantly decreased, in dual responders, at 6 h
after challenge, compared with baseline values and values at 15 min; P
< 0.05 for both comparisons (bottom right panel).
not only is required for cell adhesion and migration, but also
plays a key role in the immune response; in fact, these adhesion
molecules are involved in leukocyte functions such as antigen-
specific recognition by T lymphocytes, T lymphocyte activation,
and Ig production through T-dependent humoral immune re-
sponses (10, 13-16).
Under normal conditions, only low levels of ICAM-1 are
expressed on peripheral blood mononuclear cells, but its expres-
sion is increased upon cell activation.
In this context, our observation that ICAM-1 is upregulated
on peripheral blood T lymphocyte subsets and more markedly
on CD4' T lymphocytes, in dual asthmatic responders, suggests
that these cells are in a higher state ofactivation in DR compared
with ER and it might reflect the presence of more sustained
inflammatory changes in patients developing LAR. We can
safely exclude ICAM-1 upregulation as a feature of atopic sta-
tus, since in atopic patients with single early response, the ex-
pression of ICAM-1 on immunoregulatory T cells was not sig-
nificantly different from control subjects. The degree ofICAM-
Intercellular Adhesion Molecule-I in Dual Asthmatic Responders
1843

Page 5

35Q:
300-
250'
LU
200-
150
100~
15min
After challenge
6h
Baseline
Figure 3. Changes in serum levels of soluble ICAM-1 (s-ICAM-l) in
single asthmatic responders (n = 6) and dual asthmatic responders (n
= 6) after allergen inhalation challenge. Serum levels of s-ICAM-l,
expressed as ng/ml, are on the vertical axis; the time-points studied are
on the horizontal axis. Data are expressed as mean±SEM. Serum levels
of s-ICAM-l significantly increased, in dual responders, at 6 h after
challenge, compared with baseline values and values at 15 min; P < 0.03
for both comparisons.
1 expression, at baseline, on both CD4+ and CD8+ T lympho-
cytes from the overall asthmatic population was related to the
change in FEVI at the time of the LAR, suggesting that ICAM-
1 upregulation on immunoregulatory T cells is related to the
occurrence of the LAR. However, further studies including a
larger number of patients are needed in order to define whether
these changes can predict the occurrence of LAR.
The late phase asthmatic response is associated with airway
inflammation (25-27) and is characterized by an inflammatory
cell infiltrate in the bronchial mucosa and submucosa (28). The
upregulation ofICAM-1 on immunoregulatory T cells in periph-
eral blood might be the consequence of cytokine release from
activated airway inflammatory cells or it might reflect a more
prolonged antigenic stimulation in patients with dual asthmatic
response. As an alternative explanation, it might be the result of
a "spill-over" of activated cells from the airways into vascular
compartments. Regardless, the upregulation of ICAM-1 on T
cells may play an important role in enhancing and maintaining
airway inflammation in patients with LAR. In fact, increased
interactions between these cells and antigen-presenting cells or
other lymphocytes could lead to chronic inflammation mediated
by immunocompetent cell activation and cytokine release.
After allergen challenge, we observed a decrease of CD8+
ICAM-l+ T lymphocytes in all dual asthmatic responders, and
of CD4+ ICAM-1+ T lymphocytes in four out of six dual asth-
matic responders, at the time of the LAR. The decrease of
ICAM-1 expression on T lymphocyte subsets might be the con-
sequence ofICAM- 1 shedding from activated cells, as the solu-
ble form of ICAM-1 increases in serum of DR at the time of
the LAR. This latter observation is consistent with the results
of previous studies showing that atopic patients undergoing
segmental bronchoprovocation with antigen have s-ICAM-1
present in bronchoalveolar lavage fluid only during the late
response (43). A significant elevation of circulating s-ICAM-l
was also shown in patients with acute asthma when compared
to stable asthmatics and control subjects (34). As an alternative
explanation, the decrease ofCD8' ICAM-1+ and CD4' ICAM-
1+ T lymphocytes at the time of the LAR might reflect the
migration of activated T cells from the vascular compartment
into the airways; this latter possibility seems less likely since
both the numbers and the percentages of CD8' and CD4' T
lymphocytes did not change after allergen inhalation challenge.
However, as our study was performed only on peripheral blood
we cannot exclude the possibility that the decrease of both
CD8' and CD4' T lymphocytes expressing ICAM-1 reflects
the migration of these cells into the airways.
In this study we did not observe any change in both the
absolute number and the percentage of CD4' or CD8' T lym-
phocytes after allergen challenge in single early and dual asth-
matic responders. These results are consistent with those ob-
tained from Frew et al. in sensitized animals challenged with
aerosolized ovalbumin (44). Our findings, however, differ from
those of Gonzales et al. (30) and of Gerblich et al. (29). The
first group ofinvestigators, in fact, found that allergen inhalation
by subjects exhibiting a single early response was associated
with a significant elevation in the percentage, but not in the
absolute number ofCD4' T lymphocytes in the blood. Gerblich
et al., in contrast, showed a decrease in peripheral blood CD4'
T lymphocytes in atopic asthmatic patients after allergen chal-
lenge, but none of their patients showed LAR. These discrepan-
cies can probably be explained by the different study design,
particularly with respect to (a) the technique of allergen chal-
lenge, (b) the different time points studied, and (c) the different
disease severity of the population studied.
In conclusion, this study shows that, in dual asthmatic re-
sponders, ICAM-1 is upregulated on peripheral blood immuno-
regulatory T lymphocytes and particularly on CD4' T lympho-
cytes, suggesting that these cells are in a higher state of activa-
tion in dual asthmatic responders, compared with single early
responders. These results support the concept that more severe
inflammatory changes occur in patients with LAR and add fur-
ther evidence for an important role of adhesion molecules in
immune and inflammatory response in asthma.
Acknowledgments
This work was supported in part by a grant from the Ministero dell'Uni-
versita e della Ricerca Scientifica (fondi 60%).
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V. De Rose, G. Rolla, C. Bucca, P. Ghio, M. Bertoletti, P. Baderna, and E. Pozzi