Pentoxifylline therapy in HIV seropositive subjects with elevated TNF

Immnpharmacology ELSEVIER

Immunopharmacology31 ( 1995) 85-91

Pentoxifylline therapy in HIV seropositive subjects with elevated TNF Alexandra Kruse a,b Klaus Rieneck c, Mogens Kappel a,b Marianne Orholm Helle Bruunsgaard a,b Henrik Ullum a, Peter Skinh0j a, Bente Klarlund Pedersen a,b,*

a,

a Department of Infectious Diseases, M7721, Tagenscej 20, 2200 Copenhagen N, Denmark b The Copenhagen Muscle Research Centre, Copenhagen, Denmark c Laboratory of Medical Immunology, Department of Medicine ITA, Rigshospitalet, National Unit,ersiO' Hospital Copenhagen, Denmark

Received 3 April 1995; accepted 25 July 1995

Abstract Tumor necrosis factor-a (TNF-a) is thought to induce cachexia in subjects infected with human immunodeficiency virus (HIV), and it has been suggested that HIV-seropositive patients would benefit from treatment with pentoxifylline, a known suppressor of TNF-a production. The purpose of the present study was to examine how pentoxifylline at a dose of 800 mg thrice daily would influence the cellular immune system in HIV-seropositive persons with elevated TNF-a. Six HIVseropositive subjects with elevated amounts of TNF-a in plasma at least at two occasions were included in an open, controlled, randomized, cross-over study consisting of a 6 week treatment period and a 6 week control period. Blood samples were collected before and at the end of each period. Pentoxifylline treatment did not influence the concentration of plasma-TNF-a, subpopulations of blood mononuclear cells, the proliferative responses nor the natural killer (NK), and lymphokine activated killer (LAK) cell activities. Furthermore, pentoxifylline treatment did not influence the weight, temperature, well being, or tiredness of the subjects. However, the patients frequently reported gastrointestinal side effects. In vitro, however, pentoxifylline at suprapharmacological concentrations inhibited the blood mononuclear cell (BMNC) proliferative responses, NK, and LAK cell activities. Keywords." Pentoxifylline; HIV; AIDS; Tumor necrosis factor; Cytokine; Natural killer cell; Lymphokine activated killer cell: Lymphocyte;

Proliferative response

1. Introduction Increased levels of tumor necrosis factor-a (TNFc~)-cachectin have been found in patients infected

* Corresponding author.

with human immunodeficiency virus type 1 (HIV-1) [5,11,13]. TNF-o~ induces replication of HIV through activation of the transcription factor NF-kB, which binds to the viral promotor gene [7,9,17,19]. T N F - a may contribute to the AIDS-related wasting syndrome, since loss of weight has occurred in rats constantly infused with TNF-o~ [2] and mice with TNF-secreting tumor cells [16], and injection of

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A. Kruse et a l . / Immunopharmacology 31 (1995) 85-91

TNF-a into rats caused release of branched-chain amino acids from skeletal muscles [15]. Furthermore, TNF-a inhibits the antiretro-viral effect of Zidovudine in cell culture [12]. Pentoxifylline (Trental, Copenhagen, Denmark) was originally used to treat claudicatio intermittence, but has recently been shown also to decrease the level of TNF-a protein, and mRNA in vitro [8,10,20], and in vivo [6]. In a recent pilot study, including patients with AIDS-related wasting, pentoxifylline had no clinical effect [14]. The concentrations of pentoxifylline required in order to obtain significant reduction of TNF-c~-production in vitro 30-500 /zM [8,10,20] is not obtained during in vivo treatment with pentoxifylline. The dosage normally used is 1200 mg daily, which gives a concentration in plasma of pentoxifylline 5-10 /zM [23]. Therefore, we doubted whether even higher concentrations of pentoxifylline as used in this study (2400 mg daily) would influence the TNF-a production. However, in theory pentoxifylline might influence the TNF-o~ production indirectly, e.g. through an inhibitory effect on HIV replication. Furthermore, findings of short term in vitro culturing might not be directly transferred to long term in vivo treatment, and the present study was therefore designed to examine the effect of pentoxifylline treatment on TNF-o~ production. Unpublished data from our laboratory have shown that pentoxifylline in vitro in suprapharmacological concentrations influenced the proliferative responses, and cytotoxic activities of lymphocytes. Therefore, the effects of pentoxifylline treatment on BMNC subpopulations, NK, and LAK cell activity, and proliferative responses were investigated. In addition data on weight, temperature, tiredness, and well-being were recorded during the open, controlled, randomized, cross-over study consisting of a 6 week treatment period, and a 6 week control period.

mean CD4 counts were 263 (range 0-1247). 58 of the patients had detectable amounts of TNF-a in plasma (two times above background value). 29 of these accepted to have another blood sample taken. 17 had still elevated plasma-TNF-a. 14 were included in the project, while 3 were too weak and were excluded. 6 patients completed the study. 4 patients dropped out because of gastrointestinal side effects during the treatment period, and 4 dropped out due to poor compliance. Among the 6 patients who fulfilled the trial, none had AIDS, 4 were males, median age was 41 (range 31-50 years), their median CD4 count was 229 (8-640), 2 were treated with DDI, none received zidovudine, glucocorticoids, chemotherapy, antihypertensiva, anticoagulantia or prior treatment with pentoxifylline. They had had no acute infections two weeks prior to the study, no change in medicine within the last month, no anemia, and no sign of hemorrhagic disease (the latter diagnosis is a contraindication to treatment with pentoxifylline).

2.2. Study design We used an open, controlled, cross-over design consisting of a 6-week treatment period (a) and a 6-week control period (b). Treatment consisted of pentoxifylline (Trental, Hoechst), 800 mg thrice daily for 6 weeks. One patient had gastrointestinal side effects, and his dose was reduced to 400 mg thrice daily for the last 3 weeks. Blood samples were collected and the weight of the patients were measured before, and at the end of each 6 week period before morning medication. Each day in the 12 week study the patients measured their weight, rectal temperature, and gave a score from 1 to 5 indicating tiredness (1 = no tiredness, 5 = very tired), and well-being (1 = very well, 5 = very bad).

2.3. Isolation of blood mononuclear cells (BMNC) 2. Subjects, materials and methods

2.1. Subjects 277 consecutive HIV-seropositive patients had a blood sample taken, 78 had AIDS, 244 were males, their average age was 40 (range 16-66 years), their

BMNC were isolated by density gradient centrifugation (Lymphoprep Nyegaard, Oslo, Norway) on LeucoSep tubes (Greiner, Frickenhausen, Germany), and washed three times in medium RPMI (1640 Gibco, Grand Island, NY., USA). Cells were frozen in freezing medium (50% RPMI, 30% fetal calf

A. Kruse et al. /Immunopharmacology 31 (1995) 85-91

serum and 20% DMSO) and kept in liquid nitrogen until thawed for analyzing. BMNC were incubated in medium with or without the presence of pentoxifylline. Several concentrations of pentoxifylline were examined, but pentoxifylline (Hoechst) 8 0 / x g / m l = 280/zM induced the maximal effect on the cytotoxic activities. This concentration was therefore applied in the assays described below. 2.4. Determination o f N K cell activity

The NK cell activity was measured using K562 target cells in a SlCr release assay as previously described [18]. Effector cells were BMNC incubated with (1) medium, (2) 0.1 I U / m l IFN-a (kindly provided by Dr Robert Jordal, The Bloodbank, Copenhagen County Hospital, Gentofte, Denmark), (3) 20 I U / m l IL-2, 1 /xg/ml (Boehringer Mannheim, Germany) or (4) 280 /zM pentoxifylline, for 1 h, 37°C. Triplicates of 100 /zl mononuclear cells (5 x 106 cells/ml) and 100/zl target cells (105 cells/ml) were incubated in microtitre plates for 4 h, 37°C. The plates were centrifuged for 10 min; 100 /xl supernatant were transferred to new tubes and radioactivity was determined. Spontaneous release was determined by incubation of 100 /xl target cells with 100 /xl medium and maximum release was determined by incubation of 100 /xl target cells plus 100 /.d medium with 10% Triton X-100. Percentage sl Cr release (NK cell activity) was determined by:

87

2.6. Proliferation assay

BMNC proliferation assay was performed as previously described in details [22]. Cell cultures were performed in triplicates in microtitre plates (Nunc, Roskilde, Denmark), 6.6 X 105 BMNC were resuspended in 200 /~1 and for 72 h incubated with either (1) medium, (2) phytohaemagglutinin (PHA), 3 /zg/ml (Difco Laboratories, Detroit, MI., USA), (3) purified derivative of tuberculin (PPD), 10 /zg/ml (State Seruminstitute, Copenhagen, Denmark) or (4) IL-2 20 U / m l (Boeringer Mannheim, Germany). These incubations [[1-4]] were performed with or without the presence of pentoxyfylline 280 /xM. During the last 24 h the cells were exposed to [3H]thymidine. The cultures were collected on glass fibre filters with a harvesting machine (Skatron, Lierbyen, Norway), and the [3H]thymidine incorporation was measured in a liquid scintillation counter (TRI-CARB Packard Instruments, Rockville, MD, USA). For each triplicate the mean was recorded. 2.7. Clinical chemical tests

and was given as mean of triplicates.

Haemoglobin, MCV, leukocyte, lymphocyte and neutrophil concentrations were determined using a Cell Counter (Technicon H.1, Miles Inc., Tarrytown, NY, USA). p24 HIV antigen was detected by a sandwich-type immunoassay that used murine monoclonal antibody (anti-HIV core antigen) coated onto microwell strips (Coulter). Serum-/32-microglobulin concentrations were measured with a quantitative competitive enzyme (Pharmacia Diagnostics, Piscataway, New Jersey, USA).

2.5. Determination of L A K cell activity

2.8. FACS analysis

BMNC were incubated with IL-2 6000 I U / m l (Proleukin, Eurocetus B.V., Amsterdam, Netherlands) with or without the presence of pentoxifylline 280 /zM, 48 h, 37°C. LAK activity was measured in a SlCr release assay using DAUDI target cells. 100 /zl LAK cell suspension in concentration 1 X 10 6 cells/ml and 100 /xl target cells in concentration 2 x 104 cells/ml were added to each well in microtitre plates. From this point the assay and calculations were performed exactly as described for the NK cell assay.

Phycoerythrin-conjugated monoclonal antibodies Leu-M3 (CD14), L e u l l (CD16), Leul9 (CD56) (Beckton-Dickinson, Oxnard, CA., USA) and phycoerythrin-conjugated T4 (CD4) (Dako, Glostrup, Denmark) and fluorescein-conjugated monoclonal antibodies T8 (CD8), T3 (CD3), CD19, (Dako, Glostrup, Denmark) and Leull (CD16) (Beckton-Dickinson) were used. Thawed mononuclear cells were washed twice in phosphate-buffered saline (PBS) with 2% fetal calf serum (FCS). 1.0 X l0 s cells were resuspended in 100 /zl PBS containing FCS and incubated

( test - spontaneous) cpm %lysis =

( maximum - spontaneous) cpm

x 100

88

A. Kruse et al. / lmmunopharmacology 31 (1995) 85-91

30 minutes in 5°C with 10 /xl of one or two monoclonal antibodies. Labelled cells were washed 3 times and analyzed by flow cytometry using a fluorescence-activated cell sorter analyzer (FACS-STAR, Beckton-Dickinson). 2.9. Detection of TNF-t~ in plasma

TNF-a were detected by the commercially available ELISA-kit (Biokine, T Cell diagnostic, Cambridge, MA, USA). Inter assay coefficient of variation was 7.4%, detection limit was 10 pg/ml. 2.10. Detection of TNF-a-MRNA

RNA was purified and then measured by hybridization. RNA was purified from frozen cell pellets using the method of Chomczynski et al. [2]. Total RNA was run in 1% agarose/formaldehyde gels as described [24]. Fractionated RNA was blotted overnight onto Zeta-probe membrane (Bio-Rad, R0dovre, Denmark) in 10 SSC (1.5 M sodiumchloride, 0.15 M tri-sodium citrate, pH 7.0). The membrane was washed 5 min in 2SSC, RNA was fixed to membraqnes by UV radiation from UV transilluminator for 2 min and dried. The membrane was prehybridized for 1 h at 65°C in 10 ml hybridization buffer (1 mM EDTA, 7% sodium dodecylsulphate (SDS), 0.5 M NaHPO, pH 7.2) Hybridizations were performed in a Hybaid oven (Hybaid, Middlesex, England) overnight at 65°C by addition of 100 ng heat denatured probe previously labelled with c~32_p dCTP (Amersham, Birkercd, Denmark) using the hexamer priming according to standard protocols [4] TNF-a (EcoRI insert) was a generous gift from Dr. Arjun Singh (Genetech Inc., South San Francisco, CA, USA) and beta-actin (BamHI insert) was a generous gift from Dr. K Ryg~ird (Retsmedicinsk Institut, Copenhagen, Denmark). Plasmids were transformed into HB101, and cDNA inserts were purified according to standard methods [24], using either spun columns (Millipore, T~strup, Denmark) or Qiaex DNA extractionkit (Qiagen, Kebo, Herlev, Denmark), subsequent to electrophoretic separation in 2% agarose gels of plasmids digested with the proper restriction enzymes, After hybridization, the membrane was washed at 65°C 2 times for 30 min in 1 mM EDTA, 7% SDS, 40 mM Nail PO, pH 7.2, and

2 times for 30 minutes in 1 mM EDTA, 1% SDS, 40 mM NaH2PO4, pH 7.2. The hybridized membrane was exposed to Hyperfilm-MP (Amersham) at - 8 0 ° C using intensifying screens and developed in Kodak RP X-OMAT automatic film processor. For subsequent hybridizations the membrane was stripped by washing 2 times in 0.1 SSC at 95°C for 20 rain, the stripped membrane was dried and kept at - 20°C. 2.11. Statistical analysis

Results are expressed as means and standard error of means (SEM). Possible statistical significant differences between the two periods for any immune parameter were estimated by comparing the deltavalues for each period ( b e f o r e - after)pentoxifylline vs. (before - after)controt. A paired t-test was used, p < 0.05 was considered significant. Effect of in vitro pentoxifylline on any immune parameter was estimated by pooling all data obtained for a given immune parameter with or without pentoxifylline. Data with versus without pentoxifylline in vitro were compared. A paired t-test was used, p < 0.05 was considered significant.

3. Results 3.1. Effects of pentoxifylline in vivo

It appears from Table 1 that pentoxifylline did not change the level of plasma TNF-a concentration. Furthermore, mRNA for TNF-a in BMNC was detected in only very small amounts and did not change during pentoxifylline therapy (data not shown). Pentoxifylline did not change the percentual distribution or total concentration of BMNC subpopulations, Table 1, the NK cell activity (4 h assay) either unstimulated or stimulated with IFN-a, IL-2, or pentoxifylline in vitro, nor the LAK cell activity, Table 2. Furthermore, pentoxifylline did not change the NK cell activity (24 h assay, n = 2), or the proliferative responses, either unstimulated, or stimulated with PHA, IL-2, or PPD (n = 4). Pentoxifylline had no influence on HIV antigen, /3 2-mikroglobulin, lymphocyte concentration, Table 1, hemoglobin or neutrophil concentrations (data not shown). It appears from Table 3 that pentoxifylline did not induce

A. Kruse et al./ Immunopharmacology 31 (1995) 85-91 Table 1 Effects of six weeks treatment with pentoxifylline in vivo on plasma TNF-ot (pg/ml) percentages of CD4 + , CD8 + , CD3 + ,CD16+, CD56+, CD14+, and CD19+ BMNC and total lymphocyte concentration (109 cells/l), HIV p24 antigen, /32-microglobulin concentration (mean + SEM) Before

After treatment After control period period

Plasma TNF-o~ 18.5 + 2.5 %CD4+ cells 18.6+6.0 %CD8 + cells 40.3 + 2.6 %CD3 + cells 61.0 + 7.8 %CD16+ cells 5.7+2.4 %CD56 + cells 2.7+0.8 %CD14+ cells 9.8+3.0 %CD19+ cells 5.3+1.0 Lymphocyte 1.3+0.3 concentration HIVp24antigen 203 +130 fl2-microglobulin 212 +12

43.2 + 15.1 17.3+7.8 32.4 + 2.9 57.3 + 10.0 5.5+2.9 4.0+1.8 8.7+3.5 2.8+0.7 1.4+0.3 228 +121 261 +47

42.6 + 18.5 16.8+4.4 35.2 + 5.3 63.8 + 8.3 6.8+2.2 4.5+1.5 10.1+3.2 4.5+1.1 1.4+0.3

89

Table 4 The in vitro effects of PTX (pentoxifylline), 280 /xM/ml, on natural (NK) cell activity, 4 hours assay, NK cell activity, 24 hours assay, lymphokine activated killer (LAK) cell activity, and blood mononuclear cell (BMNC) proliferative responses (prol), either unstimulated (unstim), or stimulated (stim) with interleukin 2 (IL-2), phytohaemagglutinin (PHA), and purified derivate of tuberculin (PPD), (mean + SEM) - PTX

+ PTX

p values

NK activity, 4 h 10.7 + 2.3 9.5 + 2.5 NK activity, 24 h 50.4 + 10.7 39.9 + 9.1 LAK activity 31.7 + 7.7 17.1 + 2.7 BMNC prol, unstim 13.2 + 1.9 11.4 + 1.2 BMNC prol, IL-2 stim. 196 +56 43 + 10 BMNC prol, PHA stim. 3128 + 1020 2250 +870.0 BMNC prol, PPD stim. 27 +5 16 + 2

0.663 0.019 0.034 0.373 0.014 0.122 0.002

388 +278 245 +23 changes

in w e i g h t

(neither patients own

measure-

m e n t , n o r w e i g h t o b t a i n e d in t h e c l i n i c ) , o r t e m p e r a Table 2 Effects of six weeks treatment with pentoxifylline in vivo on natural killer (NK) cell activity (%), either unstimulated (unstim. NK), or stimulated with interleukin-2 (NK+IL-2), interferon-ct (NK+ IFN-a) or pentoxifylline in vitro (NK+PTX) and lymphokine activated killer (LAK) cell activity with or without the presence of pentoxifylline in vitro (mean + SEM)

Unstim. NK N K + IL-2 N K + IFN-o~ N K + PTX LAK LAK + PTX

Before

After treatment period

After control period

12.8+4.1 18.7+6.9 16.0+4.3 7.8+2.1 29.5+11.8 17.9+4.2

9.2+3.1 14.5+7.4 13.6+5.2 13.6+6.7 36.0+13.7 18.5+0.8

9.9+5.0 17.1+7.2 16.9+6.9 7.8+4.1 29.7+19.3 15.0+8.1

ture. T h e p a t i e n t s r e p o r t e d s l i g h t l y m o r e t i r e d n e s s , and less well-being during the treatment period, Table 3. F u r t h e r m o r e , t h e y c o m p l a i n e d a b o u t s t o m a c h p a i n , loss of appetite,

nausea,

vomiting,

and

diarrhoea

during the treatment period.

3.2. Effect o f pentoxifylline in L~itro

It a p p e a r s f r o m T a b l e 4, that p e n t o x i f y l l i n e in v i t r o ( 2 8 0 / z M ) s i g n i f i c a n t l y d e c r e a s e d t h e N K cell a c t i v i t y ( 2 4 h a s s a y ) , L A K cell a c t i v i t y a n d I L - 2 a n d PPD-stimulated proliferative responses.

Table 3 Clinical symptoms were recorded daily by the patients. The daily average weight (kg), temperature (C) and score 1 to 5 for well-being (1 = very well, 5 = very bad) and tiredness (1 = no tiredness, 5 = very tired) are shown for the treament period, and the control period. In addition weight of the patients in end of each period, measured in the clinic, is shown (mean + SEM)

Weight, measured by the patient (kg) Weight, measured in the clinic (kg) Rectal temperature, measured in the morning by the patient (C) Wellbeing Tiredness

Before

After 6 weeks treatment with pentoxifylline

After 6 weeks control period

63.1 + 4.2 64.8 + 3.4 37 + 0.1

62.9 + 0.3 64.1 + 3.4 36.8 + 0.1

63.2 + 0.2 64.7 + 3.5 36.9 + 0.0

2.8 +0.3 2.7+0.1

3 +0.I 3 +0.1

2 +0.1 2 +0.1

90

A. Kruse et aL / Immunopharmacology 31 (1995) 85-91

4. Discussion In this study we did not find any effect of pentoxifylline treatment in HIV-seropositive patients with elevated amounts of TNF-a in plasma, neither on the TNF-a protein, or mRNA in BMNC, nor on the percentual distribution of subpopulations, proliferation, or cytotoxicity of lymphocytes. Furthermore, pentoxifylline treatment did not influence the clinical symptoms recorded by the patients during the project. This is in contrast to the findings in other studies showing that pentoxifylline inhibits the TNFa level in the blood and mRNA in BMNC (reviewed by Dezube [4]). We found elevated plasma-TNF-a in around 20% of HIV-seropositive subjects. This is in accordance with another study [13]. By re-testing the subjects with detectable TNF-a in plasma only around 50% had elevated plasma-TNF-a. In the control period the concentration of plasma-TNF-a also changed. This variation was more than 100% and was not due assay variation (the coefficient of variation was 7.4%), but reflects the fact that plasma-TNF-a changes over time. Thus, the changes in plasmaTNF-a exceeded the so-called pentoxifylline-induced changes in plasma-TNF-a reported by others [4], and the data therefore show that in HIV-seropositive subjects, the concentration of plasma-TNF-a changes over time independently of pentoxifylline treatment. As found by others [25] pentoxifylline 2400 mg daily was not well tolerated, because of gastrointestial side effects. The high dose of pentoxifylline was chosen to strengthen the possibility of finding an effect of pentoxifylline treatment on the plasma-concentrations of TNF-a. The plasma-concentrations of pentoxifylline thereby achieved were still much lower than required for inhibition of TNF-a production in vitro. In theory, in vitro studies do not exclude the possibility that lower concentrations of pentoxifylline in long term administration, as obtained during treatment, may influence the TNF-a production. However, our results clearly show that even high doses of pentoxifylline in vivo neither decrease the level of plasma-TNF-a, nor influence the proliferative responses, or non-MHC restricted cytotoxic functions of lymphocytes. The latter could be expected since suprapharmacological concentrations in

vitro inhibit the proliferative responses, and cytotoxic activities. It is known that TNF-/3 is involved in the induction of cellular cytotoxicity [21]. Both the TNF-a and TNF-/3 production is inhibited by pentoxifylline [20]. The finding that pentoxifylline inhibited the NK cell activity when present in a 24 h assay, but not in a 4 h assay indicates that higher levels of TNF-/3 is produced during the cytotoxicity mediated in a 24 h assay, alternatively that TNF-a is involved in the cytolytic process. The results also suggest that TNF-a or TNF-fl are involved in IL-2 and PPD-induced proliferation. However, it was not the purpose of this study to investigate the mechanisms by which pentoxifylline inhibited the proliferative responses, lymphokine activated killer and natural killer cell activities. In conclusion, this study shows that pentoxifylline at a dose of 800 mg thrice daily does not change the plasma levels of TNF-a, and does not influence the percentages of lymphocyte subpopulations, lymphocyte proliferation or cytotoxicity. Furthermore, no signs of clinical improvement were recorded, but most subjects had gastrointestinal side effects during pentoxifylline treatment. Thus, this study does not support any role for pentoxifylline in the treatment of HIV-seropositive persons.

Acknowledgements The excellent technical assistance of Ruth Rousing and Hanne Willumsen is acknowledged. The study was supported by the AIDS Research Foundation, and The Danish National Research Foundation #504-14.

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