INTRODUCTION

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Tumor necrosis factor-alpha (TNF-), a cytokine produced mainly by activated monocytes and macrophages, plays a key role as a mediator of inflammation and cellular immune responses (1). Lipopolysaccharide (LPS) is the most potent stimulus of TNF- production. However, other bacterial cell wall components, viruses, fungi, parasites, and tumor cell membrane components can also induce its secretion from monocytes/macrophages. In the lung it is suggested that TNF- modulates granulomatous and fibrotic processes (2).

The production of TNF- by mononuclear phagocytes is regulated by the intracellular levels of cyclic adenosine monophosphate (cAMP) (3). Exogenous cAMP analogues and substances, such as prostaglandin E₂ capable of increasing the intracellular level of cAMP, reduce the release of bioactive TNF- by downregulating the expression of the TNF- gene (3, 4). Recent studies reported the ability of theophylline and pentoxifylline (POF), both phosphodiesterase inhibitors, to suppress the monocyte/macrophage TNF- production by increasing the intracellular accumulation of cAMP (5, 6).

POF is a xanthine which has been used clinically for the treatment of vascular diseases since 1984 (7). There is increasing evidence that POF may also play a therapeutic role in the inhibition of inflammatory processes. POF has been shown to improve resistance against sepsis or endotoxin challenge in mice, rats, and humans (9), most likely by decreasing circulating TNF levels (15, 16). POF is able to inhibit the synthesis of messenger RNA (mRNA) for TNF- in mouse peritoneal macrophages at the transcriptional level (8, 17). Also in humans, POF is able to reduce the release of TNF- by peripheral blood monocytes (PBM) (18). Compared with other new agents, POF is relatively inexpensive and has few side effects. A recent study suggests that patients with progressive sarcoidosis respond to POF treatment (19). This beneficial clinical effect may be due to the interference of the drug with TNF- production from alveolar macrophages (AM), because these cells frequently release increased amounts of TNF- in patients with sarcoidosis (20).

Therefore, the aim of this study was to investigate whether AM are also susceptible to POF-induced suppression of LPS-stimulated TNF- production, and how the magnitude of suppression compares with that seen in PBM. Further, we studied whether POF is able to suppress the spontaneous production of TNF- from AM in patients with active pulmonary sarcoidosis in vitro.

	METHODS

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Study Population

For comparison of PBM and AM, seven patients (2 female, 5 male) with a mean age of 40 yr (range, 26 to 55 yr) were recruited. Two of them were smokers. They underwent diagnostic bronchoscopy for various reasons (coin lesions or hilar abnormalities on chest radiography, suspicion of recurrent tuberculosis which was not confirmed). There was no evidence of interstitial lung disease. The lung lavaged was radiographically normal. Bronchoalveolar lavage (BAL) cell differentials were normal. None of them had recent therapy or other concomitant illnesses.

For investigating the effect of POF on the spontaneous TNF- production by AM, six patients with active pulmonary sarcoidosis (3 female, 3 male) with a mean age of 46 yr (range, 31 to 64 yr) were studied. Two of them were smokers. The diagnosis of active sarcoidosis was based on clinical and radiological findings along with biopsy evidence of noncaseating epithelioid-cell granulomas (23). One patient had stage I and 5 had stage II disease. None of the patients received therapy at the time of investigation. Informed consent was obtained from all patients according to institutional guidelines.

BAL Procedure

BAL was performed in a subsegment of the right middle lobe or lingula. Ten aliquots of 20 ml of sterile saline were instilled and immediately recovered by suction. The fluid was filtered through two layers of gauze and separated from cellular compounds by centrifugation (500 × g for 10 min at 4° C). Cell viability was assessed by trypan blue exclusion. The total cell number was determined and cytocentrifuge slides of BAL cells were stained with May-Grünwald-Giemsa for cell differentiation.

Cell Cultures

PBM were isolated from heparinized blood by ficoll density centrifugation, AM were collected by BAL as previously described. Cell cultures were performed as previously described (24). After three washings with phosphate-buffered saline (PBS), the PBM or AM were resuspended at a concentration of 1 × 10⁶ cells/ml in RPMI 1640 medium supplemented with 2% heat-inativated fetal calf serum (FCS), 2 mM L-glutamine, 200 U/ml penicillin, and 200 µg/ml streptomycin. From this 1 × 10⁶ cells per well were seeded in 24-well tissue culture plates and incubated at 37° C in a 5% CO₂ humidified atmosphere for 1 h. The AM and PBM were purified by adherence to the plastic culture plates. After the adherence period of 1 h, the nonadherent cells were removed by three washings with culture medium, and fresh supplemented RPMI 1640 medium was added. The adherent populations were incubated for 24 h in supplement RPMI medium alone, in medium and LPS (1 µg/ml), and in medium and LPS (1 µg/ml) with POF at concentrations of 1 mM, 0.1 mM, and 0.01 mM. The AM from the six patients with sarcoidosis were incubated for 24 h in supplemented RPMI medium alone and in medium with POF at concentrations of 1 mM and 0.01 mM. At the end of the incubation period, the culture supernatants were harvested, rendered cell-free by centrifugation, and stored at 80° C until assayed for TNF-.

Assay for TNF-

The concentrations of TNF- in culture supernatants were measured using an enzyme-linked immunosorbent assay (ELISA) kit with precoated plates (Biozol, Eching, Germany). The detection limit was 5 pg/ ml. The TNF- concentration was expressed as pg/ml/10⁶ AMs or PBMs to correct for the number of total AM or PBM.

Statistical Analysis

The data are expressed as mean ± SEM. Within each group the data were analyzed using Kruskal-Wallis test. p Values < 0.05 were considered to be significant.

	RESULTS

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Effect of POF on LPS-stimulated TNF- Production from PBM

The spontaneous TNF- production from PBM was low (155 ± 59 pg/ml/10⁶ cells). LPS induced a highly significant and more than 10-fold increase in TNF- production (1,914 ± 282 pg/ml/10⁶ cells), p < 0.01. Incubation of cells with POF showed a dose-dependent inhibition of the LPS-induced TNF- production (Figure 1A). At 0.1 mM POF the suppression was 43% (1,032 ± 230 pg/ml/10⁶ cells) of the LPS-induced TNF- production; 1 mM POF showed complete inhibition (126 ± 36 pg/ml/10⁶ cells).

View larger version (15K): [in this window] [in a new window]   View larger version (16K): [in this window] [in a new window]   Figure 1.   Inhibition of LPS-stimulated TNF- production from human mononuclear phagocytes by various POF concentrations. (A) Blood monocytes. (B) Alveolar macrophages.

Effect of POF on LPS-Stimulated TNF- Production from AM

The spontaneous TNF- production from AM was low (204 ± 69 pg/ml/10⁶ cells), and similar to that from PBM. LPS induced a highly significant and huge increase in TNF- production (6,084 ± 1,843 pg/ml/10⁶ cells), p < 0.01. Incubation of cells with POF resulted in a dose-dependent inhibition of the LPS-induced TNF- production (Figure 1B). At 0.1 mM POF, the suppression was 53% (2,842 ± 1,377 pg/ml/10⁶ cells) of the LPS-induced stimulation, at 1 mM POF the inhibition was 86%, and thus almost complete (873 ± 329 pg/ml/10⁶ cells).

Effect of POF on the Spontaneous TNF- Production from AM in Sarcoidosis

The spontaneous TNF- production was 973 ± 589 pg/ml/10⁶ cells and thus markedly higher than in nonsarcoid patients (5-fold). Incubation with POF suppressed this production almost completely at both concentrations in each individual patient (Figure 2). The TNF- production was reduced to 91% of the spontaneous production at 0.1 mM POF and to 98% at 1 mM POF.

View larger version (12K): [in this window] [in a new window]   Figure 2.   Inhibition of spontaneous TNF- production from AM in sarcoidosis by various POF concentrations.

	DISCUSSION

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The present study shows that the dose-dependent POF- induced suppression of LPS-stimulated TNF- production was not different for human PBM and AM. We also demonstrated that POF is able to inhibit the spontaneous TNF- production from AM of patients with sarcoidosis in vitro.

Our findings on human PBM confirm previous reports demonstrating the ability of POF to suppress LPS-stimulated TNF- production (6, 18). In addition our study showed that human AM from patients without interstitial lung disease respond in a similar way as PBM to the suppressive effect of POF on LPS-induced TNF- secretion. Previous data demonstrated that prostaglandin E₂ and dexamethasone treatment of normal PBM suppressed LPS-induced TNF- production by 72 to 78% whereas the same treatment suppressed TNF- production from normal AM less efficiently (only 22 to 33% suppression), suggesting differential susceptibility of these two members of the monocyte/macrophage lineage to TNF- regulation by immunomodulators (22). POF seems to suppress LPS-induced TNF- synthesis at points in the signaling pathway different from those affected by dexamethasone (25) without affecting interleukin-6 (IL-6) production (8, 13, 26). Our results showed that the inhibitory activity of POF on LPS-stimulated TNF- production by normal human PBM and AM in vitro is approximately of the same magnitude. Thus human PBM and AM are equally susceptible to the suppression of TNF- production by POF. The lowest effective concentration of POF in our in vitro study (0.1 mM) is comparable to peak therapeutic plasma concentrations of 0.1 to 0.01 mM (27). Other studies which investigated a comparable POF effect used higher concentrations than required in vivo (28, 29).

Our results in patients with sarcoidosis confirm that AM of patients with active disease spontaneously release increased amounts of TNF- (20, 21, 24, 30). This exaggerated spontaneous AM-derived TNF- production in active sarcoidosis can be almost completely inhibited by POF at therapeutic concentrations, as demonstrated in this study. Because TNF- is a central mediator for granuloma formation, POF may be a potent immunosuppressive agent in sarcoidosis. Furthermore, there is evidence for the interference of POF with T helper cells type 1 (Th1)-cytokines (31, 32) which are thought to play an important role in the pathogenesis of sarcoidosis (20, 24). Along these lines, a recent study demonstrated that interleukin-12 (IL-12) production by stimulated human blood mononuclear cells can also be suppressed by POF (33). Interestingly IL-12 is spontaneously produced by AM in sarcoidosis (34) and is important in the Th1 immune respose. Along with the suppression of the spontaneous TNF- production, inhibition of IL-12 production could be another important mechanism for the effectiveness of POF in sarcoidosis. The beneficial clinical effect of POF therapy observed in a recently published open trial (19) may thus be explained in part by reduced secretion of these cytokines from AM. A suppression of IL-2 and interferon- production by Th1 lymphocytes may also be a possible explanation, as suggested by a recent in vitro study with PBM cells (31). The expression of IL-2 receptors on human lymphocytes is also inhibited by POF (35). However, the full details of the mechanisms why POF may be a promising immunosuppressive agent in sarcoidosis have not been elaborated yet.

In conclusion, beside inhibiting LPS-induced TNF- production from PBM and AM to a similar extent, POF is also able to suppress the spontaneous TNF- production from AM in sarcoidosis in vitro. This may be the basis for further investigation of the role for POF as immunotherapeutic agent in the treatment of sarcoidosis.