Morphine-induced macrophage apoptosis: the role of transforming growth factor-beta

Abstract

Laboratory and clinical reports indicate that opiate addicts are prone to infections. This effect of opiates is partly attributed to opiate-induced macrophage (Mphi) apoptosis. In the present study, we evaluated the role of transforming growth factor-beta (TGF-beta) in morphine-induced apoptosis of murine J774 cells and peritoneal Mphi. Mphi harvested from morphine-treated mice showed greater (P < 0. 0001) apoptosis when compared with control Mphi. Morphine also enhanced apoptosis of J774 cells and peritoneal Mphi. Anti-TGF-beta antibody inhibited (P < 0.001) the morphine-induced apoptosis in J774 cells (control 0.7 +/- 0.4%; 10-6 M morphine 23.5 +/- 0.7%; anti-TGF-beta antibody (Ab) + 10-6 M morphine 8.1 +/- 0.7%; apoptotic cells/field) and peritoneal Mphi (control 1.5 +/- 0.9%; 10-6 M morphine 29.1 +/- 1.4%; 10-6 M morphine + anti-TGF-beta Ab 19. 1 +/- 1.8%; apoptotic cells/field). TGF-beta enhanced (P < 0.001) apoptosis of J774 cells and peritoneal Mphi. TGF-beta also promoted Mphi DNA fragmentation into integer multiples of 180 bp (ladder pattern). Immunocytochemical studies revealed that morphine enhanced the Mphi cytoplasmic content of TGF-beta. In addition, Western blotting showed increased production of TGF-beta by morphine-treated J774 cells when compared with control cells. Morphine increased J774 cell expression of bax. Interestingly, morphine-induced bax expression was inhibited by anti-TGF-beta Ab. As both morphine-induced J774 cell apoptosis and bax expression were inhibited by anti-TGF-beta Ab, it appears that morphine-induced J774 cell apoptosis may be mediated through the generation of TGF-beta.

Figure 1

A composite photomicrograph of J774 cell staining for apoptosis and transforming growth factor-β (TGF-β). (a), (b) and (c) J774 cells stained with H-33342 and propidium iodide. Control (a), 10⁻⁶ m morphine (b) and 10⁻⁴ m morphine (c) treated J774 cells. Apoptotic and necrotic J774 cells showed bright and pink fluorescence, respectively. J774 cells labelled for TGF-β are shown (d), (e) and (f). Control (d), 10⁻⁸ m morphine (e) and 10⁻⁶ m (f) treated J774 cells showed cytoplasmic staining for TGF-β (brown). (Magnification × 150.)

Figure 2

Effect of transforming growth factor-β (TGF-β) antibody (Ab) on morphine-induced J774 cell apoptosis. Equal numbers of J774 cells were incubated in media containing vehicle (control), morphine (10⁻⁶ m), anti-TGF-β Ab (5 μg/ml), or anti-TGF-β Ab (5 μg/ml) + morphine (10⁻⁶ m) for 24 hr. At the end of the incubation period, cells were stained with H-33342 and propidium iodide. The percentages of live and apoptotic cells were recorded. Results (mean ± SEM) are from four sets of experiments, each carried out in triplicate. *P < 0·001 compared with control, anti-TGF Ab, and anti-TGF Ab + morphine.

Figure 3

Representative DNA gel showing the effect of anti-transforming growth factor-β (TGF-β) antibody (Ab) on morphine-induced J774 cell DNA fragmentation. Equal numbers of J774 cells were incubated in media containing vehicle (control), morphine (10⁻⁶ m), anti-TGF-β Ab (5 μg/ml), or anti-TGF-β Ab + morphine (10⁻⁶ m) for 24 hr. At the end of the incubation period, cells were harvested, DNA was extracted and electrophoresed. Morphine enhanced J774 cell DNA fragmentation (lane 3), whereas control (lane 2) and anti-TGF-β Ab (lane 4) treated J774 cells did not show any DNA fragmentation. Anti-TGF-β Ab inhibited morphine-induced J774 cell DNA fragmentation (lane 5). Lane 1: molecular-weight marker.

Figure 4

Effect of morphine on the production of transforming growth factor-β (TGF-β) from J774 cells. Equal numbers of J774 cells were incubated for 24 hr in media containing vehicle (control) or different concentrations of morphine (10⁻¹⁰−10⁻⁸ m). At the end of the incubation period, cells were lysed, protein was extracted, separated by electrophoresis and probed for TGF-β production. Morphine-treated J774 cells showed increased production of TGF-β.

Figure 5

Effect of transforming growth factor-β (TGF-β) on J774 cell apoptosis. Equal numbers of J774 cells were incubated for 24 hr in media containing vehicle (control) or different concentrations of TGF-β (10 or 25 ng/ml). At the end of the incubation period, cells were harvested, DNA extracted and separated by electrophoresis. TGF-β-treated cells showed a classic ladder pattern (lanes 3 and 4). Control J774 cells did not show any DNA fragmentation (lane 2). Lanes 1 and 5: molecular-weight markers.

Figure 6

Effect of anti-transforming growth factor-β (TGF-β) antibody (Ab) on morphine-induced J774 cell expression of bax. Equal numbers of J774 cells were incubated for 24 hr in media containing vehicle (control), 10⁻⁸ m morphine, 10⁻⁶ m morphine, anti-TGF Ab (5 μg/ml), anti-TGF-β Ab + 10⁻⁸ m morphine, or anti-TGF-β Ab + 10⁻⁶ m morphine. At the end of the incubation period, cells were harvested, protein was extracted and Western blots were generated and probed with anti-bax antibody. Morphine enhanced the expression of bax. Anti-TGF-β antibody attenuated the morphine-induced J774 cell bax expression.