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357
WORKSHOP REPORT
16. Elias JA, Jimcnel. SA, Frcu ndHch B . - Recombi nant
gamma, alpha and beta interferon regulat ion o r human
ung libroblast proliferation. Am Rev Respir Dis, 1987, 135,
62- 65.
17. fl ughes DA , H aslam PL. - Changes in
phosphatidylglycerol in bronchoalveolar lavage .fluids from
patients with cryptogenic librosing alvcolitis. Chest, 1989, 95,
pB. Wcwers MD. Rcnnard SI. Adelberg S.
Modulation of alveolar macrophage-driven
i(e:tatinn by alternative macrophage mediators.
1986, 77. 700-708.
_ Tumor necrosis factor interacts wilh
' lntcrferons to inhibit libroblast proliferation
prostaglandi n -dependent and independent
Am Rev Respir Dis, 1988, 138, 652-658.
8~9.
Cellular immune responses in the lung of
hypersensitivity pneumonitis
G. Semenzato, L. Trentin
lavage (BAL) of hypersensitivity
{HP) patients is studied in the initial phases
In subacuLe or chronic phases the pattern
number of CD56 and CD57 cells co·exprcssing T-ccll
markers is predominant over those lacking these determinants. The pattern of expression of these markers in
controls is statistically different (fig. 1). Other markers
strictly defining natural killer cells are lacking on the
surface membrane of BAL cells r11. Thus, the alveoli lis
in HP patients is mostly represented by CD3+, CD8+,
CD57+, CD56+, CD16- non-major ltistocompatibility
complex (MHC) restricted cytotoxic lymphocytcs.
It is important to differentiate the pattern of BAL in
HP from that in other disorders known to be associated
with lymphocytosis. In sarcoidosis, tuberculosis and
berylliosis the BAL lymphocytcs express the helperrelated phenotype. BAL lymphocytes from patients with
interstitial pneumonia associated with collagen vascular
diseases, silicosis, histiocy tosis X, AIDS and
amiodarone pneumonitis express the suppressor/cytotoxic
phenotype. The presence of an alvcolitis characterized
by CD3+/CD8+/CD56+/CD57+/CD 16- phenotype
suggests HP. Introduction of more specific reagents may
report we will summarize the work that
in our laboratory on cell suspensions
from the BAL of HP patients.
recovery from BAL of HP patients is fivefold
and mostly represented by lymphocytes
ww,•v&•"uo evaluation demonstrated that few BAL
express B-cell related markers, most being
by T-lymphocytes [2). Analysis of subsets
COS+ lymphocytcs are the predominant
hence the CD4/CD8 ratio is extremely
0.5 vs 1.8 controls). AJthough the % of
the proUfcrarion associated markers (T9 and
s) is low, a significant difference with
10 controls exists in absolute numbers. An
of lymphocytes bearing I-ll.A-DR determinants
demonstrated.
tw.J'IfnrTn~rt
1. - Phenotypic analysis of cytotoxic cells recovered from the BAL of HP patients
CD 57
%
mlxl()l
%
TCR~1
CD16
CD56
ml x 103
%
ml
X
IQ)
%
ml
X
103
31.2
±3.7
122.5
±28.6
40.1
±9.4
88.0
±17.1
5.4
±1.5
12.8
±4.9
3.14
:W.25
5.31
±1.3
9.7
± 1.5
1.0
±0.2
4.9
±1.5
0.5
±0.1
5.1
±1.6
0.5
±0.1
1.34
±0.21
0. 13
±0.16
<0.001
<0.001
<0.001
<0.00 1
<0.00 1
<0 .001
<0.001
NS
HNK. J; CD56: N901; CDI6: NK-15; BAL: bronchoalveolar lavage; HP: hypersensitivity pneumonitis.
Pllttcm of reactivity with monoclonal antibodies
ceUs with cytotoxic phenotype shows a signifinumber of cells positive for HNK-1
and NKH-1 (CD56) reagents in the lavage of
with respect to controls (table l) (1]. The
··-Cl!n'
=- -
School of Medicine, Dept of Qioical Medicine, 1st
Padua, Italy.
•c:. 35128
help tO differentiate some of the above disorders.
Since the lung contains a separate compartment for
cytotoxic ceJJs that react to foreign antigens , the
presence of cytotoxic lymphocytcs in the BAL of HP
patients may fit with the pathogenesis of this disease.
nowbly scnsitization via the upper respiratory tract by
extrinsic antigens.
358
G. SBM£NZATO, L. TRENTIN
To clarify the structures involved in recognition of
putative antigens, we evaluated the distribution in BAL
ofT-ceiJs expressing the membrane products of the alpha/
· bel.a or gamma/delta chain products of the T-cell receptor. We found a slightly increased % of gamma/delta
posjtive ceUs. However, the absolute number of these
cells in the BAL fluid, mdicated a marked increase in the
lung of HP patients {table l). It has been proposed that
gam ma/delta T-eell receptor positive lymphocytes
display non-MHC restricted cytotoxicity; this fits well
with other phenotypic markers and functional studies
performed on BAL HP cells reported below.
Hypersensitivity
pneumonitis
Controls
0 COS7•'C03+
• COS7•1C03·
0 CO;&.ICOJ•
• C05 6•1CD3·
0 C01 6·1COJ.
•
COtG•ICOl·
• CD3•tYo •
0 COl•'Y6 ·
Fig. l. - Distribution of subpopulations or cells bearing the phenotype
of non-MHC restricted lymphocytes in the BAl. of controls and
patients with HP. Non-MHC: non-major histocompatibility comp.lex.
The number of alveolar macrophages bearing Class I
and 11 molecules (in particular DQ molecules) was
increased in HP patients, and the number of alveolar
macrophages expressi ng transferrin receptors was
decreased [3]. The observation that a higher number of
alveolar macrophages from HP patients display Class I
HLA A, B and C and Class II HLA DQ antigens is
relevant to lhe immunopathology of HP. Since the role
of Class I and Il MHC antigen in the lytic function of
cytotoxic/suppressor lymphocytes has recently been
emphasized, their involvement in recruitment of the
CD8 population is possible.
To rule out the possibility that the lung T
HP patients proliferate as a clone,
carried out LO define the clonality of ...... 11an1~,
tions. BAL T-lymphocytes were invcs1
molecular level by evaluating the T-ceU
rearrangement. The configuration of the
gamma-gene region did not show
rearrangcments, supporting the notion that
ing with a polyclonal expansion of T-ceUs
trast, the evaluation of the beta-gene region
receptor revealed frunt as novel bands in
(unpublished observations). This pattern is
for HP since a similar configuration of the
tor beta-chrun was found in sarcoidosis
Using a Pokeweed Mitogen-induc
differentiation assay, lung T-cells from HP
shown LO display a suppressor in vitro
This fi nding offers major clues to tl1c
pattern of HP. Evidence has been accu
mechanisms leading to granuloma formation
lated by the presence of regulatory T-cells.
clear cell infiltration precedes the u c•fuumm
granuloma. and the presence of different
crucial in regulating the appearance and m
granulomas, perhaps by the release of a
lymphokines. It has been demonstrated that
T -cells are correlated with an active gra
formation, whereas suppressor/cytotoxic
NK cells are associated with the regression
nomenon. Suppressor cells may slow dowrr
formation in HP patients, hence granuJomas
prominent as in other disorders, e.g. sru:-co1dOSis.•1
alveolitis is characterized by accumulation
lymphocytcs.
A significant increase of spontaneous cytotoll
observed in HP patients (1]. By contrast, BAL
cytes from asymptomatic farmers display a
vitro function superimposable on that of
difference offers an explanation for the
pathogenetic mechanisms in the two groups but
substantiated. Attempts have been made to
the nature of cytotoxic cells accounting for
patients with HP [7], and have demon
different types of cytotoxic mechanisms are
lung cells from HP patients including
non-MHC restricted T-cytotoxic and cells and
ine activated kjller cells.
The nature of soluble factors (interleukin-2
other biological response modifiers) accou ..
T-cell growth, activation and intensity of alveohus
patients needs to be determined.
In a follow-up study we subdived patients
groups, according to their (awaited/continued)
to the specific antigens [8]. At first
number of CD8+ cells with a reversal of the
rruio was seen in patients with HP.
alions showed a persistent increase of CD8"
reversal of the CD4/CD8 ratio in patients who
tO be regu larly exposed. lrrcspective or
persistent increase of cytotoxic cells was •
Cytotoxic cells showed a persistently enhanced ut
WORKSHOP REPORT
during the entire follow-up in patients who
be regularly exposed. Patients who continagricultural en~runents but witho~t ~er
specific anugens at work exh1b1ted a
CP4+ cells, a decrease rn CD8+ cells, and
of CD4/CD8 ratio to the nonnal range six
the first observation [8J.
~iqtoJIORIIcat analysis. at fli'St evaluation, showed
... u.... .... _n of lung parenchyma by CD8+ cells
~hl!eOI~cut immunohistological observations
f!CU:ii~'J""' CD8+ infiltrate in the group of
continued to ~ regularly exposed, and
of CD4+ cells after 6 months in the lungs of
were not.
indicate that alveolitis in RP patients is a
and its intensity might be modulated by
to relevant antigens, amount of antigen
of sensitization, thus explaining the
BAL lymphocytosis observed in patients
exposed to the specific antigens compared
are noL Increased cells with supprcssor/cyin the lung of these patients is
related to a local immunological response
. These mechanisms may be relevant in
the pathogenesis of HP.
Acbtowkdg•merrts: We wish to thank Drs. G.
Marcer, A. Cipria.ni for allowing to study their
patients.
References
G, Agostini C, ZambeUo R, Trentin L, Chilosi
M arcer G, Cipriani A. - Lung T cells in
pneumonitis: phenotypic and functional
ffMWIUJI, 1986, 137, 1164-1172.
2.
359
Semenzato G, C hilosi M. Ossi E, Trentin L, Piu.olo
0, Cipriani A, Agos tini C, Zambello R, Marcer 0,
Oasparotto 0. - Bronchoal veolar lavage an d lung
his tology: compar ative analys is of inrtummatory and
immunocompe.t en t cells in patients with sarcoidosis and
hypersensitivity pneumonitis. Am Rev Respir Dis, 1985, 132,
400-404.
3. Agostini C, Trenlin L, Zambello R, Luca M, Masoiarelli
M. Cipriani A, Ma rccr G, Scmenzato G. - Pulmonary
&lveolar macrophage.s in patients with sarcoidosis and hypcrsen·
sitivity pneumonitis: charactcri.zation by monoclonal antibod·
ics. J CUn lmmUIIDI, 1987, 7, 64-70.
4. Scmonzato G, Trentin L, Zambello R, Agostini C. Marccr
G, Cipriani A, Poa R. Migone N. - Cytotoxic lymphocytcs
in the lung of patients with hypersensitivity pneumonitis.
Functional and molecular analysis. AM NY Acad Sci, 1988.
532, 447-450.
5. Zambello R, Trcntin L, Casorati
Siviero F, MGSCiarolll
M, Tornma.sini A, Agostini C. - A genotypic and phenotypic
analysis of T cells proliferating in the lung of patients with
active sarcoidosis. In: Sarcoidosis and Other G ranulomatous
Disorders. C. Grassi, G. .Rizz.ato and E. Pozzi eds, Excerpt.a
Medica, Amsterdam, 1988, pp. l87- 189.
6. Semenzato 0, Agostini C, Trentin L, Za.mbcllo R, Luca
M, Marcer a, Cipriani A. - Immunoregulation in farmer's
lung cfuea.se. Correlation between the surface phenotype and
functional evaluations at pulmonary level. C~st, 1986, 89,
l33S-13SS.
7. Semen zato G, Trentin L, Zambello R, Agosti n i
C, Cipriani A, Marcer 0. - Different types of cytolo llic
lymphocyles recovered from the lung of patients with
hypersensitivity pneumonitis. Am Rtv Rtspir Dis, 1988, 137,
70-74.
8. Trentin L, Marcer 0, Chilosi M, Zambello R. Agostini C,
Ma.sciarelli M, Bizzouo R, Gemignani C. Cipriani A, Di
Vittorio 0, Scmcnzato G. - Longitudinal study of alveolitis in
hypersensitivity pneumonitis patien ts: an immunological
cva.luation. J Allergy Clin /mmUIIDI, 1988, 82, 577- 585.
a.
ce in bronchoalveolar lavage for third type immune
reactions in hypersensitivity pneumonitis
A. Pesci, G. Bertorelli*, P.P. Daii'Aglio, G.P. Neri, D. Olivieri
complex disease and immune cellular
are thought to participate in the pathogeneoe:rseru:it
pnewnonitis (HP). Data obtained
oalveolar lavage (BAL) in patients with
lung and relating to type Ill immune
in the lung are discussed.
patients with HP (32 men, 48.9±9.9 yrs)
Only two were smokers; none had previ·
treated; all had recently been exposed to the
time lapse from last exposure: 15 days).
were 7 healthy nonsmoking volunteers.
was based on standard criteria: I) history of
~~ [Q
deUe Malattie deU'Apparato Re!piratorio, Via G.
00 PARMA, It.aly.
exposure to HP antigens; 2) symptomatic acute episode
with chills, fever, cough and breathlessness 4-8 h after
exposure; 3) radiological features and/or functional
pnl!erns of intcrstjtial lung disease; 4) evidence of
antibodies against Micropo/yspora faeni.
BAL was performed after local anaesthesia ( I]. A
fibreoptic bronchoscope was wedged in a segment of the
right lobe or Lingula and a total of ISO ml of sterile 0.9%
saline (warmed to 37°C) was injected in 50 ml aliquots
with immediate vacuum aspiration. BAL fluid was
immediately filtered through two layers of surgical gauze
and the volume measured. To separate cellular and
non-cellular components, the Ouid was centrifuged (800
rpm for 10 min) and washed twice with phosphate