Investigation of the mechanism of action of nonablative pulsed-dye laser therapy in photorejuvenation and inflammatory acne vulgaris.

BACKGROUND: Nonablative lasers are widely used for treatment of wrinkles, atrophic scars and acne. These lasers stimulate dermal remodelling and collagen production, but the early molecular stimulus for this is unknown. The mechanism of nonablative lasers in inflammatory acne is variously suggested to be damage either to sebaceous glands or to Propionibacterium acnes. Their effects on cytokine production are unknown. OBJECTIVES: To assess the in vivo effects of a short pulse duration nonablative pulsed-dye laser (NA-PDL) previously used for photorejuvenation and treatment of acne, on cytokine production, P. acnes colonization density and sebum excretion rate (SER). METHODS: We examined the effect of NA-PDL (NliteV; Chromogenex Light Technologies, Llanelli, U.K.) on P. acnes colonization before and after laser therapy using a scrub-wash technique and culture at 0 and 24 h (n = 15), on SER using absorptive tape at 0, 2, 4, 8 and 12 weeks (n = 19) and on cytokine mRNA using reverse transcription-polymerase chain reaction from skin biopsies at 0, 3 and 24 h (n = 8). Results NA-PDL had no effect on P. acnes or SER. Transforming growth factor (TGF)-beta1 mRNA increased fivefold after 24 h and 15-fold in two subjects (P = 0.012). CONCLUSIONS: TGF-beta is known to be a potent stimulus for neocollagenesis and a pivotal immunosuppressive cytokine which promotes inflammation resolution. Its upregulation by NA-PDL provides a possible unifying molecular mechanism linking stimulation of dermal remodelling in photorejuvenation with inhibition of inflammation in acne. Damage to P. acnes or sebaceous glands cannot explain the effect of this device in acne.

Suppression of granulocyte/macrophage colony-stimulating factor release from human monocytes by cyclic AMP-elevating drugs: role of interleukin-10.

1. Granulocyte/macrophage colony-stimulating factor (GM-CSF) is a pro-inflammatory cytokine secreted by cells of the monocyte/macrophage lineage and has been implicated in the pathogenesis of bronchitis and asthma. 2. In the present study we have evaluated the effect of several cyclic AMP-elevating agents on lipopolysaccharide (LPS)-induced GM-CSF release from human monocytes and the extent to which the anti-inflammatory cytokine, interleukin (IL)-10, is involved. 3. LPS evoked a concentration-dependent generation of GM-CSF from human monocytes that was inhibited, at the mRNA and protein level, by 8-Br-cyclic AMP, cholera toxin, prostaglandin E2 (PGE2) and a number of structurally dissimilar phosphodiesterase (PDE) 4 inhibitors. 4. Pre-treatment of monocytes with a concentration of an anti-IL-10 monoclonal antibody that abolished the inhibitory action of a maximally effective concentration of exogenous human recombinant IL-10, significantly augmented LPS-induced GM-CSF generation. This effect was associated with a parallel upwards displacement of the concentration-response curves that described the inhibition of GM-CSF by PGE2, 8-Br-cyclic AMP and the PDE4 inhibitor, rolipram, without significantly changing the potency of any drug. Consequently, the maximum percentage inhibition of GM-CSF release was reduced. Further experiments established that the reduction in the maximum inhibition of GM-CSF release seen in anti-IL-10-treated cells was not due to functional antagonism as rolipram, PGE2 and 8-Br-cyclic AMP were equi-effective at all concentrations of LPS studied. 5. These data indicate that cyclic AMP-elevating drugs attenuate the elaboration of GM-CSF from LPS-stimulated human monocytes by a mechanism that is not mediated via IL-10. Suppression of GM-CSF from monocytes may explain, at least in part, the efficacy of PDE4 inhibitors in clinical trials of chronic obstructive pulmonary disease.

p38 MAP kinase and MKK-1 co-operate in the generation of GM-CSF from LPS-stimulated human monocytes by an NF-kappa B-independent mechanism.

1. The extent to which the p38 mitogen-activated protein (MAP) kinase and MAP kinase kinase (MKK)-1-signalling pathways regulate the expression of granulocyte/macrophage colony-stimulating factor (GM-CSF) from LPS-stimulated human monocytes has been investigated and compared to the well studied cytokine tumour necrosis factor-alpha (TNF alpha). 2. Lipopolysaccharide (LPS) evoked a concentration-dependent generation of GM-CSF from human monocytes. Temporally, this effect was preceded by an increase in GM-CSF mRNA transcripts and abolished by actinomycin D and cycloheximide. 3. LPS-induced GM-CSF release and mRNA expression were associated with a rapid and time-dependent activation of p38 MAP kinase, ERK-1 and ERK-2. 4. The respective MKK-1 and p38 MAP kinase inhibitors, PD 098059 and SB 203580, maximally suppressed LPS-induced GM-CSF generation by >90%, indicating that both of these signalling cascades co-operate in the generation of this cytokine. 5. Electrophoretic mobility shift assays demonstrated that LPS increased nuclear factor kappa B (NF-kappa B) : DNA binding. SN50, an inhibitor of NF-kappa B translocation, abolished LPS-induced NF-kappaB : DNA binding and the elaboration of TNFalpha, a cytokine known to be regulated by NF-kappaB in monocytes. In contrast, SN50 failed to affect the release of GM-CSF from the same monocyte cultures. 6. Collectively, these results suggest that the generation of GM-CSF by LPS-stimulated human monocytes is regulated in a co-operative fashion by p38 MAP kinase- and MKK-1-dependent signalling pathways independently of the activation of NF-kappa B.

Interleukin-10 does not mediate the inhibitory effect of PDE-4 inhibitors and other cAMP-elevating drugs on lipopolysaccharide-induced tumors necrosis factor-alpha generation from human peripheral blood monocytes.

Lipopolysaccharide (LPS)-induced liver injury in mice and LPS-induced tumor necrosis factor-alpha (TNF-alpha) generation by murine macrophages and hepatocytes are suppressed markedly by agents that elevate intracellular cAMP. Phosphodiesterase (PDE)-4 inhibitors, beta 2-adrenoceptor agonists, and E-series prostaglandins also attenuate the induction of the TNF-alpha gene in human monocytes in response to bacterial LPS. The mechanism of action of cAMP is unclear, but in the mouse, is believed to involve the generation of this anti-inflammatory cytokine, interleukin-10 (IL-10). In this article, we describe the results of studies designed to determine the extent to which IL-10 contributes to the suppression of TNF-alpha generation from LPS-stimulated human monocytes evoked by 8-bromo cyclic AMP (8-Br-cAMP), rolipram, salbutamol, and prostaglandin E2 (PGE2). LPS evoked a time- and concentration-dependent generation of TNF-alpha (t1/2 = 4.5 h; EC50 = 273 pg/mL), which was inhibited by exogenous human recombinant (h) IL-10 (IC50 = 124 pg/mL), and by rolipram (EC50 = 420 nM), 8-Br-cAMP (EC50 = 77 (microM), PGE2 (EC50 = 15 nM) and salbutamol (EC50 = 20 nM). In addition, 8-Br-cAMP, PGE2; and salbutamol (but not rolipram) augmented significantly LPS-induced IL-10 production (two-to-fivefold) under identical experimental conditions. Pretreatment of monocytes with an anti-IL-10 monoclonal antibody (MAb) that abolished the inhibitory action of a maximally effective concentration of exogenous hrIL-10, failed to attenuate the inhibitory effect of rolipram, PGE2, salbutamol, and 8-Br-cAMP. Anti-IL-10 was similarly inactive when the number of monocytes seeded was increased from 0.5 to 4 x 10(6)/mL or when measurements were made at 42 h post-LPS, a time when the concentration of IL-10 released was maximal. Collectively, these data suggest that in contrast to murine hepatocytes and macrophages, IL-10 does not mediate the inhibitory effect of cAMP-elevating drugs on TNF-alpha generation from human monocytes. Although the reason for this discrepancy is unclear, we suggest that the influence of cAMP on the transcriptional regulation of the TNF-alpha gene differs between species or when monocytes have differentiated into macrophages.

Albuterol does not antagonize the inhibitory effect of dexamethasone on monocyte cytokine release.

Beta2-adrenoceptor agonists given by the inhaled route are the most effective bronchodilators known, yet high doses of these drugs may be associated with an increase in asthma mortality and morbidity. One theory for this paradox is that chronic use of beta2-adrenoceptor agonists compromises the anti-inflammatory action of glucocorticosteroids. This hypothesis derives from the ability of albuterol and fenoterol to inhibit the interaction of the glucocorticosteroid receptor (GR) with proinflammatory transcriptional activators acting on the promoter region of certain target genes that encode cytokines such as tumor necrosis factor-alpha (TNF alpha) and granulocyte/macrophage colony-stimulating factor (GM-CSF). However, the functional relevance of these results has not been formally investigated. We have tested the hypothesis that albuterol reduces the ability of dexamethasone to inhibit the generation of TNF alpha and GM-CSF from lipopolysaccharide (LPS)-stimulated human monocytes. Pretreatment of human monocytes with albuterol (1 and 100 microM) for 5 and for 180 min inhibited maximally TNF alpha generation by approximately 25%. However, regardless of the concentration of albuterol, or the time of preincubation, the inhibitory effect of dexamethasone was not significantly affected with respect to the EC50 or the maximal effect produced. Qualitatively identical data were obtained when GM-CSF release was used as an index of monocyte activation. We conclude that high concentrations of albuterol do not compromise the ability of dexamethasone to suppress the generation of TNF alpha and GM-CSF from LPS-stimulated human monocytes.

Release of granulocyte-macrophage colony stimulating factor by human cultured airway smooth muscle cells: suppression by dexamethasone.

Smooth muscle cells represent a significant percentage of the total cells in the airway but their contribution to the inflammatory response seen in airway disease has not been studied. Hence, we have looked at the release of the cytokine granulocyte-macrophage colony stimulating factor (GM-CSF) in response to bacterial lipopolysaccharide (LPS) and the pro-inflammatory cytokines interleukin-1 beta (IL-1 beta), tumour necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma). Human airway smooth muscle (HASM) cells released GM-CSF under basal conditions (45.4 +/- 13.1 pg ml-1) that was significantly enhanced by IL-1 beta and TNF alpha with a maximal effect seen at 10 ng ml-1 (1.31 +/- 0.07 ng ml-1 and 0.72 +/- 0.16 ng ml-1, respectively). In contrast, neither LPS nor IFN gamma produced a significant increase in GM-CSF release. However, HASM cells exposed to IL-1 beta, TNF alpha and IFN gamma generated more GM-CSF than that evoked by any cytokine alone (2.2 +/- 0.15 ng ml-1). The release of GM-CSF elicited by the cytokine mixture was inhibited by cycloheximide and dexamethasone. These data suggest that HASM cells might play an active part in initiating and/or perpetuating airway inflammation in addition to controlling airway calibre.

Suppression of lipopolysaccharide-induced tumor necrosis factor-alpha generation from human peripheral blood monocytes by inhibitors of phosphodiesterase 4: interaction with stimulants of adenylyl cyclase.

We assessed the role of cyclic nucleotides in modulating lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha (TNF-alpha) generation in human peripheral blood monocytes. Exposure of monocytes to LPS (3 ng/ml) evoked a delayed, time-dependent generation of TNF-alpha that reached a maximum level 5-6 hr after LPS challenge and remained constant for up to 24 hr. This effect was concentration dependent and resulted in a 20-40-fold increase in the release of TNF-alpha that was sensitive to actinomycin D and cycloheximide. Treatment of monocytes with agents reputed to activate the cAMP/cAMP-dependent protein kinase (PKA) cascade in general inhibited LPS-induced TNF-alpha generation. Thus, the beta 2-adrenoceptor agonists albuterol and procaterol partially (approximately 40%) suppressed TNF-alpha generation in a propranolol-sensitive manner. Furthermore, 8-bromo-cAMP, cholera toxin, prostaglandin E2, and a number of drugs (i.e., rolipram (ZK 62711), denbufylline (BRL 30892), Ro 20-1724, benafentrine (AH 21-132), that inhibit the phosphodiesterase (PDE) 4 isoenzyme family abolished cytokine generation. In contrast, forskolin, inhibitors of PDE3 and PDE5, and activators of soluble and particulate guanylyl cyclase were essentially inactive. Interestingly, rolipram failed to potentiate the inhibitory effect of albuterol on LPS-induced TNF-alpha biosynthesis but, paradoxically, synergized with albuterol in the generation of cAMP and in the activation of PKA. When PGE2 was used to activate adenylyl cyclase, however, rolipram potentiated cAMP accumulation, PKA activation, and inhibition of TNF-alpha generation. In contrast, forskolin did not increase the cAMP content of monocytes in the absence or presence of rolipram. Collectively, these data suggest that LPS-induced TNF-alpha generation by human peripheral blood monocytes is due to increased transcription and subsequent translation of the TNF-alpha gene and that these effects are suppressed by a range of agents that activate the cAMP/PKA cascade. However, the failure of rolipram to potentiate the inhibitory effect of albuterol and procaterol on TNF-alpha generation suggests that beta 2-adrenoceptor agonists may affect gene expression and/or post-transcriptional regulatory processes by, at least in part, a cAMP-independent mechanism(s).

Pharmacological characterization of a receptor for platelet-activating factor on guinea pig peritoneal macrophages using [3H]apafant, a selective and competitive platelet-activating factor antagonist: evidence that the noncompetitive behavior of apafant in functional studies relates to slow kinetics of dissociation.

In this paper we report the characterization of a receptor for platelet-activating factor (PAF) on guinea pig peritoneal macrophages, using a radiolabeled hydrophilic PAF antagonist, [3H] apafant. [3H]Apafant bound to intact macrophages in a concentration-dependent manner that was specific, saturable, reversible, and inhibited competitively by C18-PAF (1-O-octadecyl-2-O-acetyl-sn-glyceryl- 3-phosphocholine). Scatchard transformation and Hill analysis of these data revealed that [3H]apafant identified a homogeneous population of noninteracting sites with a pKd of 8.22 nM and a Bmax of 31,600 sites/cell. The rate at which [3H]apafant associated with (Kon = 2.9 x 10(6) M-1.min-1) and dissociated from (Koff = 0.043 min-1) intact macrophages was slow, with t1/2 values of 15 and 50 min, respectively; the kinetically derived pKd was 8.3. In competition studies C18-PAF inhibited in a biphasic manner the binding of [3H]apafant to intact macrophages, which could be resolved into high (pKi = 8.27; 60%) and low (pKi = 6.06; 40%) affinity components. In macrophage membranes, the affinity of C18-PAF (pKi = 8.48) determined from competition studies with [3H]apafant was significantly reduced (pKi = 6.95) by guanosine-5'-O-(3-thio)triphosphate, whereas the mean slope of the inhibition curves was increased from 0.470 to 0.700. Functionally, C18-PAF (10 nM to 10 microM) evoked concentration-dependent .O2- generation that was biphasic in nature. Pretreatment of macrophages with apafant antagonized in a noncompetitive manner the first phase of C18-PAF (< 100 nM)-induced respiratory burst, whereas the second component (> 1 microM C18-PAF) of this response was unaffected. It is concluded that guinea pig peritoneal macrophages express receptors for PAF for which apafant has high affinity. The biphasic competition curves obtained with C18-PAF in binding experiments and the effect of guanosine-5'-O-(3-thio)triphosphate are consistent with the hypothesis that these apafant-sensitive PAF receptors are coupled to guanine nucleotide-binding proteins and can exist in at least two guanine nucleotide-regulated conformational states. It is also suggested that the noncompetitive antagonism of PAF-induced .O2- generation by apafant may be a consequence of the slow rate at which this antagonist dissociates from PAF receptors on intact macrophages.