Thymomodulin increases HLA-DR expression by ... but not T-lymphocyte proliferation in ...

Eur Respir J
1993, 6, 102-109
Thymomodulin increases HLA-DR expression by macrophages
but not T-lymphocyte proliferation in autologous
mixed leucocyte reaction
B. Balbi*, M.T. Valle**, S. Oddera+, F. Manca**, G.A. Rossi+, L. Allegra*
Thymomodulin increases HLA-DR expression by macrophages but not T-lymphocyte
proliferation in autologous mixed leucocyte reaction. B. Balbi, M.T. Valle, S.
Oddera, F. Manca, G.A. Rossi, L. Allegra.
ABSTRACT: Thymomodulin (TMD), a thymic biological response modifier,
stimulates the release of tumour necrosis factor (TNF) and granulocytemacrophage-colony stimulating factor (GM-CSF) in macrophage-lymphocyte cultures. We investigated the effects of the cytokines released in cultures with
TMD, on the expression of human leucocyte antigen-OR (HLA-DR) antigens
by alveolar macrophages (AM) and the T-cell proliferation induced in autologous mixed leucocyte reaction (AMLR) cultures or by T-cell mitogens.
Among freshly isolated AM, 84±4% were HLA-DR positive, and this proportion was significantly reduced after 24 h cultures (60±3%, p<O.OS). In cultures
without peripheral blood (PBL) lymphocytes, TMD did not change HLA-DR expression by AM CHLA-DR+AM); whilst in the presence of autologous PBL
lymphocytes, TMD induced an increase in the proportions of HLA-DR+AM
(TMD lOO j.t.g·mt·• 79+3%, p<0.04 vs control cultures). However, TMD did not
change the ability of AM to induce T -cell proliferation in AMLR between AM
and PBL lymphocytes. In contrast, in PBL mononuclear cell cultures, TMD
induced a further increase of the cell proliferation due to the T-cell mitogens
interleukin-2 (IL-2) or phytohaemagglutinin (PHA) (p<O.OS vs each control
culture with mitogens) or anti-CD3 antibodies (p<0.03 vs control cultures).
Thus, the cytokines released in cultures with TMD enhance macrophage HLADR expression. Whilst this phenomenon is not associated with changes in the
ability of AM to stimulate T -cell proliferation, TMD is able to increase the
mitogen-induced T -cell proliferation.
Eur Respir 1., 1993, 6, 102-109.
Investigating the effects of thymomodulin, a thymic
biological response modifier, on cultures of alveolar
macrophages and of blood lymphocytes, we have
previously demonstrated that thymomodulin increases
the release of tumour necrosis factor (TNF) and of
granulocyte-macrophage-colony stimulating factor
(GM-CSF) in eo-cultures of macrophages with autologous lymphocytes, and stimulates GM-CSF production
by lymphocytes [1]. Since the cytokines modulate the
activities of inflammatory and immunocompetent cells,
the ability of thymomodulin to stimulate the release of
TNF and GM-CSF suggested that it could have other
effects on macrophage and lymphocyte functions. In
the lung, the interaction between macrophages and
lymphocytes, involving cytokine release, expression of
surface molecules and cellular proliferation, is a basic
step of each immune reaction to antigenic stimuli
[2, 3]. In this context, TNF and GM-CSF, among
their many activities, are able to modulate the expres-
* Interuniversitary Center for Lung
Diseases of Northern Italy, Milan, Italy.
** Dept of Immunology, San Martino
Hospital, Genoa, Italy. + Dept of Lung
Diseases, G. Gaslini Institute, Genoa,
Italy.
Correspondence: B. Balbi
Divisione Pneumologica
Ospedale Sant'Andrea - XIX USL
19100 La Spezia
Italy
Keywords: Alveolar macrophages
autologous mixed leucocyte reaction
granulocyte-macrophage-colony stimulating factor
human leucocyte antigen DR
thymomodulin
T -lymphocytes
tumour necrosis factor
Received April 27, 1992
Accepted after revision July 13, 1992
Supported in part by a grant from
ELLEM Sri, Industria Farmaceutica, Milan, Italy.
sion of surface molecules, including human leucocyte
antigen DR (HLA-DR) molecules [4-8] . The expression of the HLA-DR surface molecules by alveolar
macrophages is a necessary prerequisite in the process
of antigen presentation to T-lymphocytes, followed by
the proliferation of antigen specific T-cells [2, 3, 9].
With this background, the present study was designed to determine whether thymomodulin is able to
modulate the expression of HLA-DR molecules on
macrophages in human alveolar macrophage and blood
lymphocyte cultures. In addition, the ability of T-cells
to proliferate in autologous mixed leucocyte reaction
(AMLR) with alveolar macrophages, or after T-cell
mitogen stimulation was tested. The data demonstrate
that thymomodulin induces an increase in macrophage
HLA-DR expression. However, thymomodulin does
not change T-cell proliferation in AMLR, but further
increases the T-cell mitogen-dependent T-cell proliferation.
THYMOMODULIN EFFECTS ON LYMPHOCYTE-MACROPHAGE INTERACTION
Materials and methods
103
from normal volunteers were cultured without T -cell
purification, as described below.
Study population
Twenty two individuals (12 males and 10 females,
28-60 yrs of age), referred to the First Division of
Lung Diseases , San Martino Hospital, Genoa, Italy,
entered this study. These individuals underwent
fibreoptic bronchoscopy as part of the evaluation for
respiratory symptoms, and/or for chest X-ray abnormalities and were chosen according to the following
criteria: 1) nonsmokers or former smokers who
quit smoking at least one year before; 2) taking no
medication at the time of the study; 3) no known
exposure to industrial or noxious pollutants; 4) resu lts of lung function tests (total lung capacity (TLC),
vital capacity (VC), forced expiratory volume in one
second (FEY 1), diffusing capacity of the lungs for carbon-monoxide (DLco)) wi thin normal values. The final diagnosis of these individuals was: adenocarcinoma
of the lung in three cases, small cell carcinoma of the
lung in three cases, epidennoid carcinoma of the lung
in four cases, localized metastatic lung involvement
from tumours arising in other organs in three cases
and tuberculoma in one case, whilst in eight cases no
evidence of bronchial or lung disease was found. Five
normal volunteers were used to obtain only peripheral
blood non-adherent cells. Informed consent was obtained before venipuncture and bronchoalveolar lavage
(BAL) procedures.
Thymomodulin
Thymomodulin (Ellem, Corso di Porta Ticincse, 89,
Milano, Italy) is derived from calf thymus by acid
lysi filtration and lyophilization. The result is a mixture of peptides, MW less than 10 000 Da. In vitro
and in vivo studies have shown that thymomodulin is
able to regulate the maturation of T-lymphocytes, to
act on functional activities of mature B- and Tlymphocytes and other inflammatory cells [10-17].
Evaluation of the expression of su1jace HLA-DR
molecules by alveolar macrophages
In order to evaluate the percentages of alveolar
macrophages expressing HLA-DR surface molecules ,
macrophages immediately after BAL (fresh
macrophages) and macrophages after 24 h cultures
(cultured macrophages) were stained with the L243
monoclonal antibody (Becton Dicki nson) , recognizing .
a non-polymorphic determinant on HLA-DR molecu les
[ 18, 19].
Macrophage culture conditions were as
described previously L1]. Briefly, alveolar macrophages obtained by means of BAL were cultured in
RPMI -1640, 5% heat-inactivated foeta l bovine serum
(FBS) (Flow laboratories), glutamine 20 mM, penicillin 100 U·ml·1 and streptomycin 100 ~-tg·ml- in 1 ml
final volume, in 24 well plates, for 24 h with or without y-interferon (y-IFN) (100 U·ml·1) or thymomodulin
at various concentrations (1, 10, 100 ~-tg·ml- 1 ). HLADR expression data were obtained from cultures of
macrophages (3x I 06 cells·ml·1) from eight patient (five
patients with neoplastic lung involvement, three
patients without lung tumours) , or from eo-cultures of
macrophages (3x l0 6 cells·mJ · 1) , obtained from nine
patients (six from patients with neop lastic lung involvement, three from individuals without lung neoplasms), with autologous blood lymphocytes (l x l0 6
cells ·mt · 1), containing >93 % CD2+ T-Jymphocytes.
The levels of various cytokines were tested in the
supernatants of these cultures [1].
Fresh or cultured macrophages (detached from the
culture wells by gentle, repeated resuspension) were
incubated at 4 oc for 20 min in phosphate buffered saline (PBS), pH 7.4, supplemented with 10% normal
human AB serum, to reduce the binding of staining
antibodies with macrophage Fe receptors. Cells were
then washed twice at 4°C in washing medium (PBS
1% FBS) and resuspended in 20 ~-tl of washing medium; 10 ~-tl of the control, isotype matched,
nonrelevant mouse antibody fluorescein isothiocyanate
(FITC)-conjugated (Becton Dickinson) or of the L243
anti-HLA-DR FITC-conjugated monoclonal antibody
were then added. After 20 min at 4°C, cells were
washed three times with washing medium at 4°C,
fixed in 1% paraformaldehyde for 30 min, and finally
1
Collection of lung and blood mononuclear cells
Cells were obtained from the lower respiratory tract
of all 22 patients by means of BAL, which was performed in all subjects in areas free of any visible
inflammatory or neoplastic endoscopic lesion, using a
total of 100 ml in five 20 ml aliquots of 0.9% sterile
saline solution, as described previously [1, 2]. After
centrifugation, determination of viability, total and
differential cell counts, the cells were resuspended at
the desired cell density in the appropriate medium (see
cell culture sections).
Heparinized blood samples were obtained by means
of venipuncture in patients undergoing fibreoptic bronchoscopy, and in five normal volunteers. Peripheral
blood monon uc lear cells were iso lated by Ficoll
density gradient [2], washed several times in Hank's
balanced salt solution (HBSS) without Ca++ and Mg++,
resuspended in RPMI-1640 (Flow Laboratories, Irvine,
Scotland, UK), with 10% autologous plasma, and
counted. Peripheral blood mononuclear cells from
patients undergoing bronchoscopy were enriched for
T-lymphocytes by adherence onto culture dishes fl ].
The non-adherent cells were resuspended and counted·
a small portion was taken for differential counts on
cytocentrifuge preparations. The percentages of CD2+
T-Jymphocytes in the final blood non-adherent cell
suspensions, hereafter referred to as lymphocytes, were
always >93 %. Peripheral blood mononuclear cells
104
B. BALB! ET AL.
resuspended in 0.5% paraformal-dehyde until fluorescence-activated cell sorter (FACS) analysis was performed. The cells were analysed with a FACS
analyser (Becton Dickinson). The data were collected
and stored in list mode files and analysed using a
Consort 30 software (Becton Dickinson). Macrophages
were discriminated from lymphocytes according to the
characteristic scatter profile on FACS analysis [20] .
To reduce the background of autofluorescence, the
FACS photomultipliers were set so that macrophage
autofluorescence in each control sample was in the
fluorescence range of 10 1• T he proportions of
macrophages staining positive were calcul ated by subtracting the control sample value from the value obtained using the anti-HLA-DR antibody. To better
compare results obtained on different days, the logarithmic amplifiers were calibrated using fluorescent
beads (lmmunosure, Coulter Immunology, Hialeah, FL,
USA; Fluotrol-GF, Ortho Diagnostic Systems, Raritan,
NJ, USA) and the analysis was performed on identical settings.
Evaluation of the ability of thymomodulin to stimulate
T-cell proliferation in AMLR
Since macrophages, like other antigen presenting
cells, interact with the T-cell antigen receptors on Tlymphocytes through the HLA-DR surface molecules,
inducing T-cell activation and proliferation [2, 3, 21 ],
we determined whether, in AMLR between alveolar
macrophages and blood lymphocytes, thymomodulin
was able to induce changes in the in vitro proliferation of T-cells. BAL cell suspensions from five
patients undergoing fibreoptic bronchoscopy (two patients with lung cancer and three patients without
neoplastic lung involvement) containing >93% alveolar macrophages, with a viability of >95%, (trypan
blue exclusion) were used. BAL cells were washed
twice and resuspended in complete medium (RPMI1640 supplemented with glutamine 20 mM, 100
U·mJ·' penicillin, 100 IJ.g·m1· 1 streptomycin, 5% FBS
and 5 mM 2-mecaptoethanol). Autologous peripheral
blood was used as a source of T-lymphocytes. T-cells
were partially purified, as described above, by adherence. The final lymphocyte suspensions, containing
>93% CD2+ T-lymphocytes, were resuspended in
complete medium. Cultures of alveolar macrophages
(AMs) al one or of peripheral blood Jymphocytes
(PBLs) alone or AMLR cultures of macrophages (previously irradiated with 3,000 rads) with lymphocytes
were performed in duplicate, with or without
thymomodulin (1, 10 and 100 IJ.g·ml·' final concentration), in 24 well plates (Flow Laboratories, Milano,
Italy), at 37°C in 5% C02 • In AMLR cultures,
2.5xl05 AMs were present in each well; since the
stimulatory capacity of antigen presenting cells is dependent upon their numbers [2, 3], graded numbers of
lymphocytes were added to each well. Maximal response was observed with a blood lymphocyte to alveolar macrophage ratio of 4:1, and the results shown
refer to these data.
Proliferation was assessed on day 6, after the addition of 20 IJ.Ci·weJI·' of tritiated thymidine (lHTdR)
(specific activity 20 IJ.Ci·mmol-1) (New England Nuclear, Boston, MA, USA) for the last 18 h of culture.
The cells were harvested with a Skatron (Sterling, VA,
USA) cell harvester, and T-cell proliferation was assessed evaluating the 3HTdR incorporation using a ~­
counter (1209 Rakbeta, LKB Wallac, Turku, Finland).
Evaluation of the ability of thymomodulin to stimulate
T-cell proliferation in mitogen-driven peripheral blood
mononuclear cell cultures
Since most of the known effects of thymomodulin
are on lymphocytes [1, 10-12], we tested the effects
of the compound on blood mononuclear cells cultured
with different T-cell mitogens. Peripheral blood
mononuclear cells (5xl05), isolated by means of Ficoll
centrifugation of heparin ized peripheral blood from
five normal individuals, were cultured in complete
medium in 96 well plates (Flow Laboratories, Milano,
Italy) for 4 days at 37°C, in 5% C0 2• Cultures were
performed with or without: 30 U·mi·' of recombinant
human interleukin-2 (IL-2) (Cetus, Emmeryville, CA,
USA), 0.1 J..lg·ml·' of anti-CD3 monoclonal antibodies
(Ortho Diagnostic, Milano, Italy) and l J..lg·ml·1 of
phytohaemoagglutinin (PHA). In addition, all the
above cultures were performed in the presence or
absence of thymomodulin (1, 10 and 100 J..lg·ml-1).
After 3 days in culture, 3HTdR was added to each well
for the last 18 h and incorporated radioactivity was
detected as described above.
Statistical analysis
All data are presented as mean±standard error of the
mean; statistical analysis for multiple comparisons was
made with the analysis of variance (ANOV A) test and
the two-tailed Student's test. The mean values of various parameters were said to be significantly different when the probability of the differences of that
magnitude, assuming the null hypothesis to be correct,
fell below 5% (i.e. p<0.05).
Results
Characterisation of cell populations recovered by
lavage
Bronchoalveolar lavage was performed in all
subjects without complications. Visualisation of the
bronchial tree in the lavaged areas demonstrated no
evidence of inflammatory or neoplastic lesions.
The recovered fluid was sterile in all cases. The total amount of fluid recovered for each 100 ml of saline infused was 66±5 ml, and the total cells recovered
were 1 L4±1.9x106, comprised of 90±2% macrophages,
THYMOMODULJN EFFECTS ON L YMPHOCYTE-MACROPHAGE INTERACTION
6±2% lymphocytes and 1.2±0.4% neutrophils or
eosinophils. There were no differences in the amount
of fluid recovered or the total and differential cell
counts, between the patients with primary or metastatic
lung tumours or without neoplastic lung involvement
(p>0.2 each comparison) (table 1).
Macrophage HLA-DR expression immediately after
bronchoscopy and after 24 h cultures with or without
thymomodulin
Evaluation of surface HLA-DR molecules of AMs
recovered by BAL from patients undergoing bronchoscopy showed that 84±4% of the cells reacted with the
anti-HLA-DR monoclonal antibody L243 (figs 1 and 2).
105
In this context, comparing samples from patients with
neoplastic lung involvement to those without neoplastic disease, no difference was found for the proportions of HLA-DR positive macrophages (84±4 vs
84±3%, p>0.5).
When macrophages were cultured in control medium
alone, without stimuli for 24 h, 62±4% of the cells
were HLA-DR positive, a percentage significantly
lower than that observed in fresh macrophages
(p<O.OS) (fig. I ). Ln contrast, when alveolar macrophages we re cultLLred for the same amount of lime in
the presence of y-rPN. 91±2% of the cells we re 1-ILADR positi ve. a value significantly higher when compared to unstimulated cultured cells (p<0.03) (fig. L).
When I, I0 and 100 ~tg·ml· ' of thymomodulin were
added to the macrophage cultures, no significant
Table 1.
Bronchoalveolar lavage data from patients with and without
neoplastic lung involvement
Fluid recovered
Cells
% Mac % Lymph % PMN
xJ06t
mJ t
Patients with
11.0±1.4
1.3±0.3
cancer (n=13)*
65±3
91±2
6±2
Patients without
cancer (n=9)*
1.1±0.4
90±2
66±4
11.5±1.8
7±2
- - - -- --- ----- ----t: per 100 ml infused; *: see methods and results for diagnosis. p>0.2 each comparison between lhe two patient groups. Mac: macrophage; Lymph: lymphocytes;
PMN: polymorphonuclear leucocytes.
---------
en
:2
~
100
100
80
80
(f)
60
:2
<{
0
0
cf?-
60
cf?-
40
40
20
20
Fresh
c
y-IFN
TMD
1
TMD
TMD
10
100
24 h cultures
Fig. I. - Proportions of HLA-DR positive alveolar macrophages
fresh and cultured with or without -y-interferon or different conccmrnt ions of thymomodulin. A lveolur mucrophngcs o htn incd from
eight pat ients undergoing fibreopuc bronchoscopy were swincd
wilh the monoclonal amibody 1..243 ( Oectt)il Dickinson) speci ric for
HLA· DR molecules. usi ng direct i mmunofluorc~cence nnd analysed
by flnw cy to mc try F re sh : va lues ob ta ined s tai ni ng aJvco la.r
mnc rophages immediately a fter bronchoHi veolar tav:1gc. A ll other
vnl uo:~ refer to mucro phagcs c ultured to 24 h with the fo llowing
stimuli: C: com rol med ium, -y- tfN: y-intc rferon, 100 U·mf· 1; TMD
I. 10 und 100: thymumod ulin I. 10 und 100 ~l g·ml '. AMs: :llveolar
macroph:tges: HLA· DR: human lc.'ul·ocyte antigen DR.
Fresh
c
y-IFN
TMD
TMD
10
24 h cultures
1
TMD
100
Fig. 2. - Proportions of HLA-DR positive alveolar macrophages
fresh and cultured in the presence of autologous peripheral blood
lymphocytcs with t'r w ithout y-i ntcrfemn or diffuem ccmcentrations
of thy momodul in. A lveolnr mncropha~e~ t)htnined from n ine patients undergoing fibreuptic bro nchoscopy were stui ncd wi th the
monoclo nal antibody L243 (Becton Diclinson) s peci fic for HLADR molecules, using direct immunofluorescence and analysed by
flow cytometry Fresh: values obtained staining alveolar macro·
phages immediately after bronchoalveolar lavage. All other values
refer to macrophages cultured for 24 h with the appropriate
stimulis. For abbreviations see legend to figure l.
B. BALBI ET AL.
106
change in the percentages of HLA-DR positive alveolar macrophages was observed, as compared to
unstimulated macrophages (p>0.5 each comparison)
(fig. I).
Similarly, when alveolar macrophages were cultured
in control medium without stimuli with autologous peripheral blood non-adherent cells for 24 h, the proportions of HLA-DR positive macrophages decreased
significantly, as compared to fresh alveolar macrophages (60±3 vs 84±4%, p<O.OS) (fig. 2). The addition of y-IFN to the macrophage-lymphocyte cultures
increased, after 24 h, the proportions of HLA-DR positive macrophages (88±3%, p<0.04 with 24 h macrophage-lymphocyte cultures without stimuli) (fig. 2).
The addition of thymomodulin at the concentration of
1 and 10 J.Lg·m1·' did not change the percentages of
HLA-DR positive macrophages after 24 h cultures
(p>O.S with control cultures) (fig. 2).
In contrast, flow cytometric analysis clearly demonstrated that in cultures of macrophages and autologous
blood lymphocytes, the addition of 100 j..lg·ml· 1 of
thymomodulin was associated with an increase in the
proportions of HLA-DR positive macrophages (79±3%,
p<0.04 with control cultures) (fig. 2). Figure 3 depicts an example of flow cytometric analysis of HLADR expression by cultured macrophages. In this
example, alveolar macrophages, obtained through
bronchoalveolar lavage from a patient with lung cancer, were cultured with autologous peripheral blood
lymphocytes in complete medium, without (Panel A)
or with the addition of thymomodulin 100 J.Lg·ml· 1
(Panel B). Note the increased proportion of HLA-DR
positive macrophages in the culture with thymomodulin (Panel B), as compared with macrophages
cultured without thymomodulin (Panel A).
Table 2. - Proportions of HLA-OR positive alveolar
macrophages in cultures with autologous peripheral
blood lymphocytes from patients with and without
neoplastic lung involvement
Stimuli
Patients with
cancer (n=6)*
Patients without
cancer (n=3)*
-------
Control
TMD 1
TMD 10 TMD 100
61±3
57±4
65±4
79±3
59±2
58±3
64±4
78±2
*: see methods and results for diagnosis. HLA-DR positive
alveolar macrophages are expressed as percentages of total
alveolar macrophagcs. p>0.2 each comparison between two
patient groups. TMD 1, 10 and 100: thymomodulin, 1, 10
and 100 J.tg·mJ·'; HLA-DR: human Leucocyte antigen.
Throughout the study, no difference was seen
between patients with or without neoplastic lung involvement in the proportions of macrophages cultured
alone or with autologous lymphocytes (table 2) reacting with the anti-HLA-DR antibody (p>0.2, each
comparison).
Ability of thymomodulin to stimulate in vitro T-cell
proliferation in AMLR
In mixed leucocyte reaction (MLR) cultures, HLADR positive antigen presenting cells induce the in vitro
proliferation of T-lymphocytes [2, 3]. In our experiments AMs, as antigen presenting cells, were able to
induce the proliferation of autologous blood T-cells
(fig. 4). Tn this context, PBLs cultured alone showed
a minimal incorporation of 3HTdR (180±32 cpm) (fig.
4). Similarly, AMs showed no significant proliferation when cultured alone (125±15 cpm) (fig. 4).
B. WrthTMD
A. Wrthout TMD
5
5
4
4
3
3
2
2
<?
C>
x2l
c:
~
0
ci
z
Fluorescence intensity
Fig. 3. - Examples of flow cytometer fluorescence profiles of alveolar macrophages (AM) from a patient with lung cancer, cultured in
the presence of autologous peripheral blood lymphocytes. without (Panel A) or with the addition of thymomodulin 100 )lg·ml·' (Panel B).
Shown in each panel are AMs reacting with a control antibody-fluo.rescein isothiocyanate (FITC) conjugated (dotted line) and with the
L243 anti human leucocyte antigen (HLA) DR aotibody-FITC conjugated (solid line). Only cells reacting with lhe anti-HLA-DR antibody
and more fluorescent than the control sample are considered HLA-DR positive. The quantitation data for % positive macrophages shown
in figures I and 2 are based on analysis of similar data for each subject.
THYMOMODULIN EFFECTS ON LYMPHOCYTE-MACROPHAGE INTERACTION
8
7
6
<?
x
5
E
4
0
0..
u
1
I
!
107
in the MLR experiments included two patients with
neoplastic lung involvement, cell proliferation data
from the patients without lung cancer were similar to
the data obtained grouping all patients. In this regard,
MLR cultures of cells from the non-neoplastic patients
had an incorporation of 5,800±550 cpm, that was not
significantly changed in MLR cultures with thymomodulin (p>0.2) (not shown).
3
Ability of thymomodulin to stimulate in vitro T-cell
proliferation in mitogen-driven T-cell cultures
2
c
TMD 1
TMD10
TMD 100
Fig. 4. - T-cell proliferation in alveolar macrophage stimulated
autologous mixed leucocyte reaclions, wilh and wilhout
lhymomodulin (TMD). Shown are data from duplicate cultures of
alveolar macrophages (2.5x!0 5 cells) (lir); of peripheral blood
lymphocytcs, (I x!0 6) ( j ); and of the eo-cultures of alveolar
macrophages, previously irradialed with 3,000 rads, and autologous
blood lymphocyres. Cells were obtained from five individuals undergoing bronchoscopy for clinical purposes. During the last 18 h
of culture, 1HTdR was added, and proliferation was assessed by
determining the amount of 1 I-ITdR incorporated by the cells and is
expressed as cpmxi0·3 . C: control medium; TMD 1, 10 and 100:
lhymomodulin I, JO and 100 !!g·ml·'.
Table 3. - Proliferative response to different T-cell
mitogens of peripheral blood lymphocytes cultured with
and without thymomodulin
Stimuli
Control
TMD 1 Jlg·mJ·'
TMD 10 !lg·mt·•
TMD J00 J.l.g·mi-1
IL-2
30 U·mt·•
anti-CD3
0.1 Jlg·ml·1
1.8±0.5
2.9±0.2*
2.8±0.4*
3.3±0.4*
2. 1±0.3
3.6±0.3**
3.8±0.4**
4.1±0.4**
PHA
I Jlg·mt·•
22±2
26±4
37±4*
35±3*
Data are expressed as cpmxl0'3• Each mean±sEM includes
data from five normal individuals. TMD: thymomodulin; IL2: interleukin-2; PHA: phytohaemagglutinin. *: p<0.05 compared with cultures with IL-2 or with PHA but without
TMD; **: p<0.03 compared with cultures with anti-CD3
antibodies but without TMD.
When irradiated AMs were eo-cultured with purified
autologous blood lymphocytes, there was a clear stimulation ofT-cell proliferation (5,950±580 cpm, p<O.Ol
each comparison with macrophages alone and with
lymphocytes alone) (fig. 4). The addition to the MLR
cultures, as well as to the control cultures of
macrophages alone or of lymphocytes alone, of 1, 10
and 100 Jlg·ml·' of thymomodulin, was not associated
with any significant change in 3HTdR incorporation
(p>O.S each comparison) (fig. 4). Thus, despite the
ability of thymomodulin to induce a higher HLA-DR
expression by AMs, the addition of thymomodulin to
MLRs did not increase the ability of macrophages to
stimulate T-cell proliferation.
Finally, although the patient population participating
In T-lymphocyte cultures with T-cell mitogens, the
cell proliferation is basically dependent upon the ability of T-cells to be directly activated through different surface molecules. The addition of recombinant
IL-2, of anti-CD3 antibodies and of PHA to cultures
of peripheral blood mononuclear cells was associated
with increased T-cell proliferation (control cultures
0.4±0.2 cpm xl0·3, cultures with lL-2 1.8±0.5 cpm
x10·3 , cultures with anti-CD3 2.1±0.3 cpm x10·3, cultures with PHA 22±2 cpm x10· 3 , p<0.05 each comparison) (table 3). Thymomodulin at 1 J.Lg·ml- 1 was
able to further increase the T-cell proliferation induced
by the addition of recombinant IL-2 (cultures with
IL-2 and with thymomodulin 1 j..lg·ml·' 2.9±0.2 cpm
xi0·3, p<O.OS with cultures with IL-2 alone) and that
stimulated by anti-CD3 antibodies (cultures with antiCD3 and with thymomodulin 1 J..Lg·ml·' 3.6±0.3 cpm
xJ0·3 p<0.03 with cultures with anti-CD3 alone)
(table 3). Similarly, in cultures of non-adherent cells
stimulated with PHA the addition of 10 Jlg·mJ·' of
thymomodulin was associated with increased cell
proliferation (cultures with PHA and with
thymomodulin 10 Jlg·ml·' 37±4 cpm x10-3 , p<0.05
with cultures with PHA alone) . At higher
thymomodulin concentrations this effect on T-cell proliferation was plateauing for all the tested mitogens
(table 3).
Discussion
We studied the in. vitro effects of a calf thymus
derivative, thymomodulin, on alveolar macrophages
and blood lymphocytes and demonstrated that
thymomodulin: 1) increases the HLA-DR expression
by macrophages in macrophage-lymphocyte cultures;
2) does not influence the ability of macrophages
to induce autologous T-cell proliferation in mixed
leucocyte reaction; and 3) increases the T-cell proliferation induced by T-cell mitogens in blood mononuclear cell cultures.
In a study on the effects of thymomodulin on
cytokine production by macrophages and lymphocytes,
we demonstrated that thymomodulin is able to induce
an increase of the T NF and the GM-CSF levels in
macrophage-lymphocyte cultures and an increase of
the release of GM-CSF in blood lymphocyte cultures
(1). The experiments included in this paper represent
108
B. BALBI ET AL.
pan of the effort to understand the functional implications of the thymomodulin-dependent cytokine release.
As a first step, since TNF has the ability to
synergize with GM-CSF in the induction of HLA-DR
molecule expression on blood monocytes and on
mononuclear phagocytes [6, 8], we evaluated HLA-DR
antigen expression by macrophages and observed an
increased HLA-DR expression in cultures with
thymomodulin. Consistent with the fact that increased
levels of TNF and of GM-CSF were observed only
in macrophage-lymphocyte eo-cultures with thymomodulin [1], the increased HLA-DR expression was
present only when macropbages were eo-cultured with
autologous lymphocytes, and not in cultures of
macrophages alone. These data suggest that one of
the effects of the cytokines released in increased
amounts in cultures with thymomodulin is the enhancement of HLA-DR expression by macrophages.
The presence of surface HLA-DR molecules is a
necessary requirement for the ability to present antigens and to induce the proliferation of T-lymphocytes
[2, 3, 9, 21, 22]. The enhanced macrophage Class 11
molecule expression induced by thymomodulin, however, was not associated with changes in T -cell
proliferation in the autologous mixed leucocyte cultures
between alveolar macrophages and blood lymphocytes.
In this context, it has been shown that the in vitro
T-cell proliferation is not better stimulated by macrophages as compared with monocytes, despite the fact
that a higher proportion of AMs than blood monocytes
express HLA-DR surface antigens [2, 3, 9]. Therefore, it is recognised that higher HLA-DR expression
does not necessarily mean better antigen presenting
function, at least as judged by these in vitro assays.
In other systems, such as eo-cultures of HLA-DR positive bronchial epitheHal cells and of autologous Tcells, the expression of HLA-DR molecules, although
sustained by y-IFN, is not coupled with the functional
capability to stimulate autologous T -cell proliferation
in MLR [22J. This evidence suggests that these in
vitro assays might not be sensitive enough or that the
increased levels of HLA-DR expression on the surface
of a given cell population may be relevant in other
types of cellular interactions.
Thymic biological response modifiers are believed to
act primarily on lymphocyte. Consistently with this
concept, the effects of thymomodulin on other cell
types, such as on macrophage cytokine release [1] and
HLA-DR expression, are probably mediated through
the changes induced by the compound on T-ceJJs. The
increased proliferation of T-lymphocytes stimulated
with different mitogens induced by thymomodulin is
consistent with the known effects of thymic hormones
and peptides, including thymomodulin [1, 10-14, 23].
Although the presence of a primary or metastatic
carcinoma of the lung may be associated with abnormalities of cell functions of immune related cells, in
this study and in other experiments with human
ciliated bronchial epithelial cells [22], we did not find
any obvious difference between cancer and non-
cancer patients for HLA-DR expression, and T -cell
proliferation. Nevertheless, the data from this paper
do not rule out the possibility that the effects of
thymomodulin on cells, including macrophages and
lymphocytes, from patients with cancer, might be different from the effects on cells of patients without
cancer.
Finally, although some of the in vitro effects of
thymomodulin described in this paper and in a study
on cytokine production [1] are observed at concentrations of the compound considerably higher the expected in vivo tissue concentrations [ 16, 17], animal
studies suggest that a tissue concentration similar to
those that we used in the in vitro assays could be
safely reached in humans, either with a systemic or a
topical administration. This observation might suggest
the possibility of increasing the amounts of thymomodulin administered in clinical practice, at least
in selected patients.
Acknowledgements: This work was performed
as part of the Ph.D. programme of B. Balbi in
Respiratory Diseases supported by the
Interuniversitary Center of Northern Italy for
Lung Diseases and the Italian Ministry of University and Scientific and Technologic Research.
The authors are grateful to F. Trave and A. Tiri,
Ellem Sri. for their help in conducting this work.
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