Paul G. Hellewell

Phosphodiesterase (PDE)4 inhibitors: anti-inflammatory drugs of the future?

Phosphodiesterase type 4 (PDE4) plays a major role in modulating the activity of virtually all cells involved in the inflammatory process. Inhibitors of this enzyme family display impressive anti-inflammatory and disease-modifying effects in a variety of experimental models. In this review, Mauro Teixeira, Robert Gristwood, Nicola Cooper and Paul Hellewell examine the capacity of PDE4 inhibitors to exert anti-inflammatory actions in vivo and discuss the potential of this class of drugs to take their place as novel therapeutic agents for a variety of inflammatory diseases.

Tumor Necrosis Factor and Steroid Metabolism in Chronic Heart Failure: Possible Relation to Muscle Wasting

OBJECTIVES We sought to assess the possible relations between clinical severity of chronic heart failure and catabolic factors, specifically tumor necrosis factor (TNF), soluble TNF receptors 1 and 2 (sTNFR-1 and sTNFR-2), cortisol, testosterone and dehydroepiandrosterone (DHEA). BACKGROUND Chronic heart failure is associated with loss of muscle bulk that may be related to alteration of the balance between catabolism and anabolism. METHODS Sixty-three patients (average age +/- SD 60.4 +/- 11.3 years) with stable chronic heart failure and 20 control subjects aged 52.8 +/- 11.4 years were studied. We measured body mass index (BMI) and obtained maximal incremental exercise testing with metabolic gas exchange measurements and measurements of venous levels of TNF, sTNFR-1 and sTNFR-2, cortisol and DHEA. RESULTS There was no difference in total TNF-alpha levels between patients and control subjects (9.76 +/- 8.59 vs. 6.84 +/- 2.7 pg/ml). sTNFR-1 (128.9 +/- 84.5 vs. 63.6 +/- 23.3 pg/ml, p < 0.003) and sTNFR-2 (250.1 +/- 109.5 vs. 187.9 +/- 92.2 pg/ml, p = 0.03) were higher in patients. DHEA was lower in patients (9.88 +/- 6.94 vs. 15.64 +/- 8.33 nmol/liter, p = 0.004). The ratio of log cortisol to log DHEA correlated with log TNF level (r = 0.50, p < 0.001 for the patients alone; r = 0.48, p < 0.001 for the group as a whole). Peak oxygen consumption correlated with both sTNFR-1 and sTNFR-2 (r = -0.51, p < 0.001 and r = -0.39, p < 0.001, respectively). There was a negative correlation between BMI and TNF levels (r = -0.43, p < 0.001 for the patients) and the cortisol/DHEA ratio (r = -0.32, p = 0.01 for the patients). CONCLUSIONS There is an increase in TNF and its soluble receptors in chronic heart failure. This increase is associated with a rise in the cortisol/DHEA (catabolic/anabolic) ratio. These changes correlate with BMI and clinical severity of heart failure, suggesting a possible etiologic link.

Leptin Indirectly Activates Human Neutrophils via Induction of TNF-α

Leptin, the satiety hormone, appears to act as a link between nutritional status and immune function. It has been shown to elicit a number of immunoregulatory effects, including the promotion of T cell proliferative responses, and the induction of proinflammatory cytokines. Leptin deficiency is associated with an increased susceptibility to infection. As polymorphonuclear neutrophils (PMN) play a major role in innate immunity and host defense against infection, this study evaluated the influence of leptin on PMN activation. The presence of leptin receptor in human PMN was determined both at mRNA and protein levels, and the effect of leptin on PMN activation, as assessed by CD11b expression, was evaluated using flow cytometry. In contrast to monocytes, which express both the short and long forms of the leptin receptor (Ob-Ra and Ob-Rb, respectively), PMN expressed only Ob-Ra. Leptin up-regulated the expression of CD11b, an early marker of PMN activation, on PMN in whole blood, yet it had no effect on purified PMN, even those treated by submaximal doses of TNF-α or PMA. The kinetics of leptin-induced activation in whole blood were consistent with an indirect effect mediated by monocytes, and 71% of the leptin-stimulatory effect on PMN was blocked by a TNF-α inhibitor. Leptin-mediated induction of CD11b expression was observed when purified PMN were coincubated with purified monocytes. In conclusion, although leptin activates PMN, it does so indirectly via TNF-α release from monocytes. These findings provide an additional link among the obesity-derived hormone leptin, innate immune function, and infectious disease.

Alveolar Macrophage Apoptosis Contributes to Pneumococcal Clearance in a Resolving Model of Pulmonary Infection

The role of alveolar macrophages (AM) in host defense against pulmonary infection has been difficult to establish using in vivo models. This may reflect a reliance on models of fulminant infection. To establish a unique model of resolving infection, with which to address the function of AM, C57BL/6 mice received low-dose intratracheal administration of pneumococci. Administration of low doses of pneumococci produced a resolving model of pulmonary infection characterized by clearance of bacteria without features of pneumonia. AM depletion in this model significantly increased bacterial outgrowth in the lung. Interestingly, a significant increase in the number of apoptotic AM was noted with the low-dose infection as compared with mock infection. Caspase inhibition in this model decreased AM apoptosis and increased the number of bacteremic mice, indicating a novel role for caspase activation in pulmonary innate defense against pneumococci. These results suggest that AM play a key role in clearance of bacteria from the lung during subclinical infection and that induction of AM apoptosis contributes to the microbiologic host defense against pneumococci.

Chemokine-induced eosinophil recruitment. Evidence of a role for endogenous eotaxin in an in vivo allergy model in mouse skin.

Selective eosinophil recruitment into tissues is a characteristic feature of allergic diseases. Chemokines are effective leukocyte chemoattractants and may play an important role in mediating eosinophil recruitment in various allergic conditions in man. Here, we describe a novel mouse model of eosinophil recruitment in which we have compared the in vivo chemoattractant activity of different C-C chemokines. Furthermore, we describe the use of antibodies to chemokines and receptor blockade to address the endogenous mechanisms involved in eosinophil recruitment in a late-phase allergic reaction in mouse skin. Intradermal injection of mEotaxin and mMIP-1alpha, but not mMCP-1, mRANTES, mMCP-5, or mMIP-1beta, induced significant 111In-eosinophil recruitment in mouse skin. Significant 111In-eosinophil recruitment was also observed in an active cutaneous anaphylactic reaction. Pretreatment of skin sites with antieotaxin antiserum, but not an antiMIP-1alpha antibody, suppressed 111In-eosinophil recruitment in this delayed-onset allergic reaction. Similarly, desensitization of the eosinophil eotaxin receptor CCR3 with mEotaxin, or blockade of the receptor with metRANTES, significantly inhibited 111In-eosinophil recruitment in the allergic reaction. These results demonstrate an important role for endogenous eotaxin in mediating the 111In-eosinophil recruitment in allergic inflammation, and suggest that blockade of the CCR3 receptor is a valid strategy to inhibit eosinophil migration in vivo.

Pyocyanin Production by Pseudomonas aeruginosa Induces Neutrophil Apoptosis and Impairs Neutrophil-Mediated Host Defenses In Vivo

Clearance of neutrophils from inflamed sites is critical for resolution of inflammation, but pathogen-driven neutrophil apoptosis can impair host defenses. We previously showed that pyocyanin, a phenazine toxic metabolite produced by Pseudomonas aeruginosa, accelerates neutrophil apoptosis in vitro. We compared wild-type and pyocyanin-deficient strains of P. aeruginosa in a murine model of acute pneumonia. Intratracheal instillation of either strain of P. aeruginosa caused a rapid increase in bronchoalveolar lavage neutrophil counts up to 18 h after infection. In wild-type infection, neutrophil numbers then declined steadily, whereas neutrophil numbers increased up to 48 h in mice infected with pyocyanin-deficient P. aeruginosa. In keeping with these differences, pyocyanin production was associated with reduced bacterial clearance from the lungs. Neutrophil apoptosis was increased in mice infected with wild-type compared with the phenazine-deficient strain or two further strains that lack pyocyanin production, but produce other phenazines. Concentrations of potent neutrophil chemokines (MIP-2, KC) and cytokines (IL-6, IL-1β) were significantly lower in wild-type compared with phenazine-deficient strain-infected mice at 18 h. We conclude that pyocyanin production by P. aeruginosa suppresses the acute inflammatory response by pathogen-driven acceleration of neutrophil apoptosis and by reducing local inflammation, and that this is advantageous for bacterial survival.

Loss of bone mineral in patients with cachexia due to chronic heart failure

We studied body composition and cytokine levels in 58 patients with heart failure and 16 control patients using dual-energy x-ray absorptiometry. Bone mineral content and density, and lean and fat tissue content, were reduced in cachectic compared with noncachectic patients and control subjects, with negative relations between indexes of bone composition and tumor necrosis factor and tumor necrosis factor receptor 1.

A novel method for isolation of neutrophils from murine blood using negative immunomagnetic separation.

Inappropriate neutrophil activation has been implicated in the pathology of several clinically important inflammatory conditions. Although murine models are extensively used in the investigation of such pathological processes, a reliable method by which viable, quiescent neutrophils can be isolated from murine blood has not been developed. Here we describe a novel method based on negative immunomagnetic separation, which yields highly pure populations of murine neutrophils. Blood is incubated with a cocktail of antibodies against specific cell markers on unwanted cells, and then with secondary antibody-coated magnetic beads. After running the preparation through a column within a magnetic field, labeled cells are retained, and a neutrophil-rich effluent is collected. This method yields a >95% pure suspension of >97% viable neutrophils, recovering approximately 70% of neutrophils from whole blood. Flow cytometric analysis shows little difference in surface L-selectin and CD18 expression on isolated neutrophils compared with neutrophils in whole blood, indicating that neutrophils are minimally activated bythe isolation process. Stimulation with phorbol 12-myristate 13-acetate (PMA) reduced L-selectin andincreased CD18 expression. Isolated neutrophilsmigrate under agarose in response to fMLP, and fluorescently labeled neutrophils transfused into recipient mice interact with postcapillary venules in a manner comparable to endogenous leukocytes. These findings show that neutrophils isolated using this method can be used for inflammatory studies in vitro and in vivo.

Caspase-1-Deficient Mice Have Delayed Neutrophil Apoptosis and a Prolonged Inflammatory Response to Lipopolysaccharide-Induced Acute Lung Injury

Caspase-1, the prototypic caspase, is known to process the cytokines IL-1β and IL-18 to mature forms but it is unclear whether, like other caspases, it can induce apoptosis by activation of downstream protease cascades. Neutrophils are known to express caspase-1, to release IL-1β and to undergo rapid, caspase-dependent apoptosis. We examined apoptosis and IL-1β production in peripheral blood neutrophils of caspase-1-deficient and wild-type mice. Constitutive apoptosis of caspase-1-deficient neutrophils was delayed compared with wild-type neutrophils and LPS-mediated inhibition of apoptosis was absent, but caspase-1-deficient neutrophils were susceptible to Fas-mediated apoptosis. LPS-stimulated IL-1β production was absent from caspase-1-deficient neutrophils. To ascertain whether these differences in apoptosis and IL-1β production would alter the response to acute lung injury, we studied pulmonary neutrophil accumulation following intratracheal administration of LPS. Caspase-1-deficient mice showed increased, predominantly neutrophilic pulmonary inflammation, but inflammation had resolved in both wild-type and deficient animals by 72 h after LPS instillation. IL-1β production was increased in wild-type lungs but was also detected in caspase-1-deficient mice. We conclude that caspase-1 modulates apoptosis of both peripheral blood and inflammatory neutrophils, but is not essential for IL-1β production in the lung.

Role of prostaglandins and nitric oxide in acute inflammatory reactions in guinea-pig skin

1 Oedema formation in skin is dependent on a synergism between mediators that increase vascular permeability and mediators that enhance local blood flow. Leukocyte accumulation is also enhanced by mediators that increase local blood flow. In this study, we have investigated whether nitric oxide (NO), an important endogenous vasodilator, could modulate oedema formation and leukocyte accumulation in guinea‐pig skin. 2 Local administration of the NO synthesis inhibitor, NG‐nitro‐l‐arginine methyl ester (l‐NAME), dose‐dependently inhibited the oedema formation induced in response to intradermal injection of bradykinin or histamine. l‐NAME, but not NG‐nitro‐d‐arginine methyl ester (d‐NAME), also inhibited oedema formation in response to i.d. injection of platelet‐activating factor (PAF), zymosan‐activated plasma (ZAP) and in a passive cutaneous anaphylactic (PCA) reaction. 3 N‐iminoethyl‐l‐ornithine (l‐NIO) was less effective and about 100 times less potent than l‐NAME in inhibiting bradykinin‐induced oedema formation. The cyclo‐oxygenase inhibitor, ibuprofen, had little effect on oedema responses induced by bradykinin, PAF and in a PCA reaction. On the other hand, histamine‐induced oedema formation was significantly suppressed by ibuprofen. 4 The inhibition by l‐NAME of bradykinin‐induced oedema formation was reversed by co‐injection of sodium nitroprusside (SNP) or prostaglandin E1 (PGE1). 5 L‐NAME inhibited 111In‐eosinophil and 111In‐neutrophil accumulation induced by i.d. injection of ZAP. 111In‐eosinophil accumulation induced by PAF and in the PCA reaction was also inhibited by l‐NAME but not by d‐NAME. 6 Co‐injection of SNP or PGE1, reversed the inhibition by l‐NAME of ZAP‐induced oedema formation and 111In‐neutrophil accumulation. SNP, but not PGE1, also reversed the effects of l‐NAME on ZAP‐induced 111In‐eosinophil accumulation. 7 l‐NAME caused a significant decrease in basal cutaneous blood flow when injected alone or with bradykinin. Again, SNP or PGE1 reversed the effects of l‐NAME suggesting that the inhibitory action of l‐NAME on oedema formation and cell accumulation was due to an inhibition of vasodilator tone in the microcirculation. 8 Thus, it appears that in guinea‐pig skin the inhibition of the production of endogenous NO inhibits both leukocyte accumulation and oedema formation induced by different mediators of inflammation. Since administration of l‐NAME also causes a local decrease in basal blood flow, we suggest that this is the mechanism by which it exerts anti‐inflammatory effects in this model.