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.

* 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.

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-

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 103

Materials and methods

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.

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 uclear 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

from normal volunteers were cultured without T -cell purification, as described below.

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- 1 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 l06 cells·mJ ·1) , obtained from nine patients (six from patients with neoplastic lung in­volvement, three from individuals without lung neo­plasms), with autologous blood lymphocytes (l x l06

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

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 101

• The proportions of macrophages staining positive were calculated 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 (RPMI-1640 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) alone 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% C02• 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 105

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).

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 were cultLLred for the same amount of lime in the presence of y-rPN. 91±2% of the cells were 1-ILA­DR positi ve. a value significantly higher when com­pared to unstimulated cultured cells (p<0.03) (fig. L). When I, I 0 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

Patients with cancer (n=13)* Patients without cancer (n=9)*

Fluid recovered mJt

65±3

66±4

Cells xJ06t

11.0±1.4

11.5±1.8

% Mac % Lymph % PMN

91±2 6±2 1.3±0.3

90±2 7±2 1.1±0.4 - - - - - - ------------ ----- -----

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.

100

80

en 60 :2 ~

0 cf?-

40

20

Fresh c y-IFN TMD 1

TMD 10

24 h cultures

TMD 100

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. using direct i mmunofluorc~cence nnd analysed by flnw cyto mc try F resh : va lues obta ined s taining aJvcola.r mnc rophages immediately a fter bronchoHi veolar tav:1gc. A ll other vnluo:~ refer to mucro phagcs c ultured to 24 h with the fo llowing stimuli: C: comrol med ium, -y- tfN: y-intc rferon, 100 U·mf·1; TMD I. 10 und 100: thymumodulin I. 10 und 100 ~l g·ml '. AMs: :l lveolar macroph:tges: HLA· DR: human lc.'ul·ocyte antigen DR.

100

80

(f)

60 :2 <{

0 cf?-

40

20

Fresh c y-IFN TMD 1

TMD 10

24 h cultures

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 without y-i ntcrfemn or diffuem ccmcentrations of thy momodul in. Alveolnr mncropha~e~ t)htnined from n ine pa­tients undergoing fibreuptic bronchoscopy 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.

106 B. BALBI ET AL.

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).

A. Wrthout TMD 5

4 <? C>

x 3 2l c:

~ 0 2 ci z

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 Control TMD 1 TMD 10 TMD 100

Patients with cancer (n=6)* 61±3 57±4 65±4 79±3 Patients without cancer (n=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 5

4

3

2

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 L YMPHOCYTE-MACROPHAGE INTERACTION 107

8

7

I 6 1 ! <? 5 0

x E 4 0.. u

3

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!05 cells) (lir); of peripheral blood lymphocytcs, (I x!06) ( 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 1I-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

IL-2 anti-CD3 PHA Stimuli 30 U·mt·• 0.1 Jlg·ml·1 I Jlg·mt·•

Control 1.8±0.5 2.1±0.3 22±2 TMD 1 Jlg·mJ·' 2.9±0.2* 3.6±0.3** 26±4 TMD 10 !lg·mt·• 2.8±0.4* 3.8±0.4** 37±4* TMD J 00 J.l.g·mi-1 3.3±0.4* 4.1±0.4** 35±3*

Data are expressed as cpmxl0'3• Each mean±sEM includes data from five normal individuals. TMD: thymomodulin; IL-2: 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 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).

Ability of thymomodulin to stimulate in vitro T-cell proliferation in mitogen-driven T-cell cultures

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 TNF 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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