Int. J. Immunopharmac., Vol. 9. No. 4. pp. 483-488, 1987. 0192-0561/87 $3.00+ .00 Printed in Great Britain. © 1987 International Society for lmmunopharmacology.

I N VITRO I M M U N O M O D U L A T O R Y EFFECTS OF INTERLEUKIN-2 A N D THYMOSIN FRACTION V IN

ACQUIRED IMMUNE DEFICIENCY SYNDROME

MOHAN M. REDDY and MICHAEL H. GRIECO

R. A. Cooke Institute of Allergy, St. Luke's/Roosevelt Hospital Center, 428 West 59th Street, New York, NY, 10019, U.S.A.

(Received 18 August 1986 and in final form 19 December 1986)

A b s t r a c t - - A monoclonal antibody (anti-Tac) that appears to bind the receptor for interleukin-2 (IL-2) was used to quantitate lymphocytes that express IL-2 receptors (IL-2R) in response to phytohemagglutinin (PHA) stimulation. 62 _+ 4% of the cells expressed IL-2R in response to PHA in twelve normal subjects compared to 22 _+ 4% in fourteen patients with acquired immune deficiency syndrome (AIDS) (P<0.001) and 50 ___ 6% in six patients with AIDS-related complex (P<0.1). There was no effect on IL-2R expression, when lymphocytes from seven controls were incubated with IL-2 (20u/ml) or thymosin fraction V (10u/ml) for 72 h. However, when the lymphocytes from seven patients with AIDS were incubated with IL-2, the IL-2R rose from 18 + 3% to 31 + 3°70 (P<0.005) and with a fraction V to 29 _+ 3% (P<0.001). In addition, IL-2 augmented the PHA-induced proliferative responses in patients with AIDS-related complex and AIDS and normal controls, whereas thymosin fraction V had no significant effect. Thymosin fraction V also enhanced the IL-2 production of PHA-stimulated mononuclear cells obtained from six patients with AIDS and six normal controls. These results suggest that both IL-2 and thymosin fraction V can modulate in vitro T-cell function in patients with AIDS.

Character is t ic l abora to ry features of acquired i m m u n e deficiency syndrome (AIDS) include lymphopenia , cu taneous anergy, in vitro unresponsiveness to mi togens and ant igens and reversal of the rat io of T helper to suppressor lymphocytes (Fauci, 1982). In addi t ion , d iminished Tac receptor (IL-2R) expression and IL-2 p roduc t ion in response to s t imula t ion has been repor ted in pat ients with AIDS (Prince, Ke rman i -Arab & Fahey, 1984; C iobanu , Welte, Kruger, Venuta , Gold, Fe ldman, Wang, Moore , Safai & Mer te l smann , 1983; Kirkpatr ick, Davis, Horsburgh , Cohn , Penley & Judson , 1985).

In ter leukin-2 (IL-2 or T cell g rowth factor) is p roduced by T lymphocytes af ter ant igen or mi togen s t imula t ion and is required for the pro l i fe ra t ion of act ivated T cells (Welte & Mer te l smann , 1985). Mitogens or ant igens induced b o t h IL-2 p roduc t ion as well as IL-2 receptor expression s imul taneously (Welte & Mer te l smann , 1985). Thymos in f rac t ion V has been shown to increase i m m u n e responsiveness in chi ldren with p r imary immunodef ic iencies (Goldstein, Low, Zatz, Hal l & Naylor , 1983) and

also IL-2 p roduc t ion of no rma l h u m a n lymphocytes (Zatz, Oliver, Samuels, Skotnicki , Sztein & Goldste in , 1985).

In this paper , we repor t tha t (a) bo th IL-2 and thymos in f ract ion V enhanced IL-2R expression, (b) IL-2 but not f rac t ion V part ial ly restored diminished lymphocyte prol i fera t ion and (c) thymos in f ract ion V enhanced p roduc t ion of IL-2 in no rma l controls and in pat ients with AIDS.

E X P E R I M E N T A L P R O C E D U R E S

Subjects

Per iphera l b lood was ob ta ined f rom 22 n o r m a l controls , 21 pat ients with AIDS-re la ted complex and 33 pat ients with AIDS meet ing the Centers for Disease Con t ro l Surveil lance def ini t ion of AIDS. AIDS-re la ted complex refers to pat ients in impl icated risk groups with non-specif ic symptoms such as fever, weight loss, general ized persis tent l y m p h a d e n o p a t h y and chronic persis tent or in te rmi t ten t d ia r rhea which are no t o therwise explained. AIDS pat ients included seven with

483

484 M. M. REDDY and M. H. GRIECO

Kaposi's sarcoma and Pneumocystis carinii pneumonia, 20 with P. carinii pneumonia, and four complicated by one or more of the following infections: histoplasmosis (2), toxoplasmosis (2) and esophageal candidiasis (1).

Lymphocyte separation

Peripheral blood was drawn in syringes containing ten units of heparin (Sigma Chemical Co.,St. Louis, MO) per 1 ml of blood. Mononuclear cells were isolated by centrifugation over Ficoll - Hypaque and were washed three times with Hanks' Balanced Salt Solution (HBSS, GIBCO, Grand Island).

Quantitation of Tac + (IL-2R) cells

Tac +ve ceils were quantitated according to Yachie, Miyanwaki, Uwadana, Ohzeki & Taniguchi (1983) with some modification. 2 × 106 mono- nuclear cells were cultured in 2 ml of RPMI 1640 culture medium containing 10oT0 fetal calf serum without mitogen or with phytohemagglutinin (Difco Detroit, MI) (PHA) 2.5 /ag/ml concentration in 24 multiwell tissue culture plates (Flow Labs, Inc., McLean, VA) at 37°C in a humidified atmosphere of 5o70 CO2 in air for 3 days. In addition, recombinant IL-2 (Hoffman-LaRoche, Inc., Nutley, N J) was added to some wells containing PHA to give a final concentration of 20U/ml (optional concentration) and thymosin fraction 5 (kindly provided by Dr Allan Goldstein of George Washington University) to give a final concentration of 10/ag/ml (optional concentration). After culture, the cells were washed four times in RPMI medium and were tested for expression of Tac antigen as defined by monoclonal anti-Tac antibody (kindly provided by Dr T. A. Waldman of NIH). The stimulated cells were incubated with monoclonal anti-tac antibody (1:1000 dilution) at 4°C for 30 min. After washing twice with Dulbecco's phosphate buffered saline (PBS) containing 0.1o70 sodium azide, 0.2 ml of 1:40 dilution of fluorescein-labeled goat anti-mouse IgG antibody (G/M) FITC, Cappel Labs, Inc. Cochran- ville, PA) and further incubated at 4°C for 30 min, washed twice with PBS and resuspended in 0.1 ml of PBS with 30o7o glycerol. The percentage of positive cells as determined by examining at least 200 cells under Leitz fluorescent microscope.

Lymphocyte culture

Lymphocyte responses to PHA were determined by culturing 2 × 105 cells, in triplicate, suspended in 0.2 ml minimum essential medium (GIBCO) supplemented with 15o70 heat-inactivated human AB

serum, 0.25 mM Hepes buffer, 2 mM L-glutamine and 100 units/ml penicillin and 100 ug/ml streptomycin (GIBCO) in flat bottomed wells of microliter plates (Flow Labs, Inc. McLean, VA) without mitogen as well as with 2.5 /ag/ml PHA. Recombiment IL-2 was added to give a final concentration of 20 U/ml and thymosin fraction V to give a final concentration of 10 gg/ml. Cells were incubated in 5o70 CO:humidified incubator at 37°C. 3H-thymidine (New England Nuclear, Boston, MA) was added at the end of 3 days. After an overnight incubation, the cells were harvested with an automatic cell harvestor (Adaps, Inc. Dedham, MA) and counted in a Beckman liquid scintillation counter. The results were expressed in counts/min for the difference between stimulated and unstimulated cultures.

Generation of IL-2

Mononuclear cells were resuspended in RPMI 1640 medium supplemented with 25 mM Hepes buffer, 1 o70 human AB serum, gentamycin and 2 mM L-glutamine. The cells were cultured in 24 flat bottom well plates (Flow Labs, Inc., McLean, VA) at 37°C in 5o70 CO2 in air for 24 and 48 h at I x 10 6

lymphocytes per ml in 2 ml containing no PHA, PHA 2.5/ag/ml and PHA and two concentrations of thymosin fraction V (100 and 200 /ag/ml) (sub- optimal and optimal). At the end of the culture, the supernatants were collected and stored at - 7 0 ° C until assayed.

Assay for IL-2 activity

IL-2 activity of supernatants was determined by its ability to support the growth of the murine IL-2 dependent cell line CTLL-I-10 according to Flomberg, Welte, Mertelsmann, Kerman, Ciobanu, Venuta, Feldman, Kruger, Kirkpatrick, Dupont & Orielly (1983) with some modifications. Cells were washed and 0.1 ml containing 4 x 103 cells was mixed with 0.1 ml serial dilutions of the test sample in 96 well flat bottom microtiter plates. Twenty-four hours later 0.5 /a Ci of 3H-thymidine was added to each well. After 4 h, the cells were harvested and ~H- thymidine incorporation was measured in a liquid scintillation counter. Each and every assay was compared to a biological response modifiers program reference reagent, human IL-2 (JURAKAT). One reference unit was defined as the amount of IL-2 in 1 ml that will induce IL-2 dependent murine T cells to incorporate 3H-thymidine at 50o70 of their maximum level after 24 h of incubation. Data were analyzed by probit transformation for determination of the 50°70 end point (Gillis, Ferm & Smith, 1978).

IL-2 and Thymosin Fraction V in AIDS

Table 1. Frequency of PHA-stimulated lymphocytes expressing Tac antigen (IL-2 receptor)

Groups No. o7o Tac-positive P value* (mean _+ S.E.M.)

Normal subjects 12 62 _+ 4 Pts with AIDS related complex 6 50 _+ 6 P<0.1 Pts with AIDS 14 22 _+ 4 P<0.001

*According to Student's t-test.

485

Table 2. Effect of interleukin-2 and thymosin fraction V on the expression of Tac antigen on PHA-stimulated lymphocytes

Groups Subjects % Tac-positive lymphocytes (mean _+ S.E.M.) Baseline Interleukin-2 20 units/ml Thymosin FracV l0/ag/ml

Normal controls 7 61 _ 5 67 _+ 2 64 _ 4 N.S. N.S.

7 18__.3 3 1 + 3 29+_.3 <0.005 <0.001

P value* Pts with AIDS

P value

*According to Student's t-test.

Table 3. Effects of interleukin-2 and thymosin fraction V on the expression of Tac antigen on PHA-stimulated lymphocytes in seven patients with AIDS

Clinical manifestations OKT4 OKT8 ratio

% Tac-positive lymphocytes

IL-2 Thymosin Frac V Baseline 20 units/ml 10/ag/ml

Pneumocystis carinii pneumonia 0.26 18.5 27.0 Pneumocystis carinii pneumonia 0.61 17.5 34.5 Pneumocystis carinii pneumonia 1.10 11.0 26.0 Pneumocystis carinii, pneumonia and

Kaposi's sarcoma 0.66 20.0 30.0 Kaposi's sarcoma and histoplasmosis 0.22 13.0 20.0 Kaposi's sarcoma - - 32.0 39.0 Histoplasmosis 0.24 16.0 38.0

Mean 18.3 _+ 2.6 30.6 _+ 2.6

32.0 30.0 19.0

28.0 29.0 46.0 19.0

29.0 _+ 3.0

RESULTS

The mean (_+ S .E.M.) percent lymphocytes expressing IL-2 receptor (IL-2R) in response to P H A in twelve normal heterosexual men was 62 _+ 4 compared with 22 _+ 4 in fourteen patients with AIDS (Table 1). This difference is statistically significant (P<0.001). A mean o f 50 _+ 6 lymphocytes expressed IL-2R in six patients with AIDS-rela ted complex.

The effect o f IL-2 and thymosin fract ion V on the express ion o f IL-2R on P H A - s t i m u l a t e d

lymphocytes are shown in Tables 2 and 3. There was no significant effect when the lymphocytes f rom seven controls were incubated with P H A and IL-2 or thymosin fract ion V. However , when lymphocytes f rom seven patients with AIDS were incubated with IL-2, the IL-2R rose f rom 18 +_ 3 to 31 +_ 3% and with thymosin fraction V to 29 +_ 3o/o. These increases are statistically significant. The effect o f IL-2 on P H A - induced proliferative responses in controls and patients are listed in Table 4. IL-2 enhanced lymphocyte t r ans format ion f rom 79,101 _+ 5841 to

486 M. M. REDDY and M. H. GRIECO

Table 4. Effect of interleukin-2 on PHA-induced proliferative responses in patients and controls

Groups No. OKT4 OKT8 ratio

PHA-induced lymphyctyte transformation (counts/min) mean ± S.E.M.

Baseline 20 units/ml IL-2

Normal subjects 11 >1.5 79,101 _+ 5814

P value*

AIDS-related complex 14 0.8 ± 0.1 58,521 ± 10,720

P value* AIDS 13 0.4 ± 0.1 17,318 ± 4692

P value*

90,401 ± 5877 <0.001

74,074 ± 11,997 <0.001

31, 171 _ 6447 <0.001

*According to Student's t-test.

Table 5. Effects of thymosin fraction V on PHA-induced lymphocyte proliferative responses in controls and patients

Groups No. PHA-induced lymphocyte transformation (counts/min) mean + S.E.M.

Baseline Thymosin Frac V 10/~g/ml

Normal subjects 5 94,289 ± 18,548 104,823 ± 23,547 AIDS-related complex 3 70,858 + 10,079 75,882 + 12,019 AIDS 6 17,246 ± 7600 21,402 ± 7960

90,401 -+ 5877 in eleven normal controls, from 58,521 +- 10,720 to 74,074 _+ 11,997 in patients with AIDS-related complex and from 17,318 +_ 4692 to 31,171 _+ 6447 in patients with AIDS. However, thymosin fraction V had no significant effect on PHA-induced lymphocyte proliferative responses in normal controls and in patients with AIDS-related complex and AIDS (Table 5).

Table 6 shows the effect of thymosin fraction V on the production of IL-2 by peripheral blood lymphocytes obtained from six normal controls and six patients with AIDS. Thymosin fraction V enhanced 11,-2 production at both 24 h and 48 h intervals after PHA stimulation with 100/ag/ml and 200/ag/ml thymosin fraction V in normal controls. The IL-2 produced in six patients with AIDS was 0.48 _-!- 0.15 U/ml at 24 h compared to 1.02 U/ml in normal controls. This difference was statistically significant (P<0.025). At 48 h culture, 0.84 _+ 0.26 U/ml were produced by patients with AIDS compared to 5.73 _+ 1.87 U/ml in normal controls. This difference also was statistically significant. Thymosin fraction V enhanced the production of IL- 2 significantly in these patients, the enhanced levels never reached levels of normal control subjects.

DISCUSSION

Normal peripheral blood T lymphocytes upon activation by antigens, mitogens and monoclonal antibodies such as OKT3 induced both IL-2 production as well at IL-2R expression. As a result of interaction between IL-2 and its receptor, T cells proliferate (Welte & Mertelsmann, 1985).

Resting T cells do not produce IL-2 nor do they respond to IL-2. Our results agree with the results obtained by Prince et aL (1984) who reported that IL-2R expression was significantly reduced in patients with AIDS and they noticed a similar trend in patients with lymphadenopathy syndrome. It has been shown during the replication or release of HTLV I, the virion becomes preferentially associated with the Tac antigen (Lando, Sarin, Megson, Greene, Waldman, Gallo & Broder, 1983).

We used two immunomodulators, IL-2 and thymosin fraction V to see their effect on Tac receptor expression. Both enhanced significantly Tac antigen expression of lymphocytes in patietns with AIDS. IL-2 has been shown to regulate its own receptor expression and is required for their optimal expression on activated T lymphocytes (Reem & Yeh,

IL-2 and Thymosin Fraction V in AIDS 487

Table 6. Effect of thymosin fraction V on IL-2 production in six normal controls and six patients with AIDS

Time PHA 2.5/ag/ml Interleukin-2 units/ml, mean _+ S.E.M.

PHA 2.5/ag/ml thymosin PHA 2.5/ag/ml thymosin fraction V 100/ag/ml fraction V 200/ag/ml

Normal controls (n = 6) 24 1.02 __. 0.23

P value* 48 5.73 __. 1.87

P value* AIDS (n = 6)

24 0.48 __. 0.15

P value* 48 0.84 _+ 0.26

P value*

1.56 _ 0.37 1.97 _+ 0.62 <0.01 <0.05

8.22 _ 2.17 9.71 ___ 2.46 <0.001 <0.005

0.77 +__ 0.22 1.09 + 0.27 <0.025 <0.05

1.55 __. 0.41 1.70 _+ 0.91 <0.05 <0.005

*According to Student's t-test.

1984; Smith & Cantrell, 1985). The exogeneous IL-2 added probably regulated the expression of Tac antigen on T cells in patients with AIDS because these patients have been shown to produce often reduced amounts of IL-2 and IL-2 is needed for optimal expression of Tac antigen. The mechanism by which thymosin fraction V enhanced Tac antigen expression is unknown, although it has been shown to induce the differentiation of pre-T cells and modulate the function of mature T cells (Goldstein et al. , 1983).

We next investigated the effects of IL-2 and thymosin fraction V on proliferative responses of lymphocytes obtained from patients with AIDS- related complex and AIDS. IL-2 enhanced significantly and partially reconstituted the depressed lymphocyte proliferative responses in patients with AIDS-related complex and AIDS, although, thymosin fraction V had no significant effect on proliferation in these two groups.

Ciobanu et al. (1983) reported that highly purified IL-2 was able to restore, partially, lymphocyte proliferation in vitro in the presence of mitogens in patients with AIDS. Similar partial reconstitution of proliferative responses was obtained by Lifson, Benike, Mark, Kothe & Engelman (1984) who added recombinant IL-2 to lymphocytes obtained from AIDS patients which enhanced their mitogen induced proliferation. Thymosin fraction V enhanced Tac antigen in vitro in our studies but not their proliferation. The reason for this differential effect is not clear. The proliferative responses of T

cells to IL-2 was blocked selectively by low concentrations of sodium azide, while it had no effect on IL-2 production (Feldman, Mertelsmann, Venuta, Andreef, Welte & Moore, 1983). Thymosin fraction V may operate in a manner similar to sodium azide in its effects on proliferation and Tac antigen expression.

Our results also indicated that mononuclear cells obtained from patients with AIDS produce significantly lower levels of IL-2 than controls. Reduced levels of IL-2 production in AIDS patients were also observed by Ciobanu et al. (1983) and by Kirkpatrick et al. (1985). On the other hand, two published reports suggested normal production of IL-2 by mitogen-stimulated lymphocytes of patients with AIDS (Prince et al., 1984; Reuben, Hersh, Murray, Munn, Mehta & Mansell, 1985). Furthermore, our results suggest that thymosin fraction V enhanced significantly the production of IL-2 by PHA-stimulated mononuclear cells of both AIDS patients and normal subjects, although the IL- 2 levels in AIDS lymphocyte cultures never reached control levels. The reconstitution of IL-2 was only partial. Recently, Zatz et al. (1985) have reported that the thymosin fraction V increased the production of IL-2 by PHA-stimulated normal human peripheral blood lymphocytes.

In conclusion, our results confirmed the T cell defect in patients with AIDS, namely reduced Tac antigen expression, lymphocyte proliferative responses and IL-2 production. IL-2 enhanced the Tac antigen expression and lymphocyte proliferative

488 M. M. REDDY and M. H. GRIECO

responses. Thymosin fraction V on the other hand, enhanced Tac antigen expression and IL-2 production.

Acknowledgements - - The authors wish to express their appreciation to Ms Mary Moriarty for technical assistance and Ms Marion Mitchell for preparation of the manuscript.

The authors also wish to thank Dr Michael Brunda of Hoffman-LaRoche, Inc., Nutley, NJ for providing IL-2 dependent cell line, Dr Allan Goldstein for providing thymosin fraction 5, Dr T. A. Waldman for providing monoclonal anti-Tac antibody and to Dr Arthur Englard for his help in recruiting patients.

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