André-Bernard Tonnel

Increased secretion of tumor necrosis factor α and interleukin-6 by alveolar macrophages consecutive to the development of the late asthmatic reaction

The late asthmatic reaction (LAR), consecutive to bronchial allergen challenge, is characterized both by the influx of various cells in proximal and distal airways and by the enhancement of bronchial hyperresponsiveness. However, the exact conditions for the development of the inflammatory reaction during the LAR remain to be specified. Since monokines play a key role in inflammatory processes, particularly in the lung, the production of tumor necrosis factor-alpha (TNF-alpha), interleukin; 1-beta (IL-1-beta) and interleukin-6 (IL-6) by alveolar macrophages (AM), collected 18 to 20 hours after exposure to allergen, was evaluated in 15 allergic subjects with asthma submitted to a challenge test with Dermatophagoides pteronyssinus (N = 6) or with wheat flour (N = 9) and in three healthy subjects. After bronchial provocation test, four patients presented no bronchial response (group 1), and six patients, a single early reaction (group 2). In contrast, five patients developed successively an immediate plus a late response (group 3). The monokine production was compared to that from nine allergic subjects with asthma studied at baseline (group 0) and from 11 unchallenged healthy subjects (control subjects). Measurements of cytokines were evaluated for TNF-alpha and IL-1-beta by a specific immunoradiometric assay, whereas IL-6 levels were appreciated by the proliferation of 7TD1 cells. No detectable amounts of TNF-alpha, IL-1-beta, and IL-6 were in bronchial alveolar lavage fluid, even after a tenfold concentration. In contrast, a significant increase of TNF-alpha (10,642 +/- 3127 U/ml) and IL-6 (1250 +/- 427 U/ml) concentrations was noted in AM supernatants from patients exhibiting an LAR (group 3) compared to cells recovered from groups 2, 1, and 0 and to challenged or unchallenged control subjects (805 +/- 244, 995 +/- 521, 1269 +/- 524, 688 +/- 85, and 445 +/- 74 pg of TNF-alpha per milliliter, respectively; 190 +/- 64, 114 +/- 91, 242 +/- 95, 80 +/- 9, and 54 +/- 19 U/ml of IL-6 per milliliter, respectively). No modification of IL-1-beta contents could be detected between the different groups. A significant correlation was detected between concentrations of TNF and IL-6 (r = 0.92; p less than 0.001). These results demonstrate TNF-alpha and IL-6 secretion by AM consecutively to the development of LAR in allergic subjects with asthma, confirming that AMs are activated after allergen challenge.(ABSTRACT TRUNCATED AT 400 WORDS)

ESM-1 Is a Novel Human Endothelial Cell-specific Molecule Expressed in Lung and Regulated by Cytokines

We here report the identification of a novel human endothelial cell-specific molecule (called ESM-1) cloned from a human umbilical vein endothelial cell (HUVEC) cDNA library. Constitutive ESM-1 gene expression (as demonstrated by Northern blot and reverse transcription-polymerase chain reaction analysis) was found in HUVECs but not in the other human cell lines tested. The cDNA sequence contains an open reading frame of 552 nucleotides and a 1398-nucleotide 3′-untranslated region including several domains involved in mRNA instability and five putative polyadenylation consensus sequences. The deduced 184-amino acid sequence defines a cysteine-rich protein with a functional NH2-terminal hydrophobic signal sequence. Searches in several data bases confirmed the unique identity of this sequence. A rabbit immune serum raised against the 14-kDa COOH-terminal peptide of ESM-1 immunoprecipitated a 20-kDa protein only in ESM-1-transfected COS cells. Immunoblotting and immunoprecipitation of HUVEC lysates revealed a specific 20-kDa band corresponding to ESM-1. In addition, constitutive ESM-1 gene expression was shown to be tissue-restricted to the human lung. Southern blot analysis suggests that a single gene encodes ESM-1. A time-dependent up-regulation of ESM-1 mRNA was seen after addition of tumor necrosis factor α (TNFα) or interleukin (IL)-1β but not with IL-4 or interferon γ (IFNγ) alone. In addition, when IFNγ was combined with TNFα, IFNγ inhibited the TNFα-induced increase of ESM-1 mRNA level. These data suggest that ESM-1 may have potent implications in the areas of vascular cell biology and human lung physiology.

Pulmonary Embolism in Patients with Unexplained Exacerbation of Chronic Obstructive Pulmonary Disease: Prevalence and Risk Factors

Context Pulmonary embolism (PE) is common in patients with chronic obstructive pulmonary disease (COPD) exacerbations, and the 2 conditions present similarly. Content For 45 months, every patient presenting with severe COPD exacerbation of unknown cause received an evaluation for PE that included a spiral computed tomography scan and color Doppler ultrasonography of the legs. Twenty-five percent of 197 patients had PE. Malignant disease, history of thromboembolism, and a decrease in Paco 2 level relative to baseline were the only factors associated with PE. Cautions This was a single-center study. Implications We need additional studies to confirm the high prevalence of PE in unexplained severe exacerbations of COPD and to study the value of routine testing for PE in patients with this clinical presentation. The Editors The management of patients with suspected acute pulmonary embolism (PE) has greatly improved in recent years because of clinical assessment of the probability of PE, pretest probability, ultrasonography, ventilationperfusion scanning, and spiral computed tomography angiography (CTA) (1, 2). However, clinical diagnosis of acute PE is difficult in patients with chronic obstructive pulmonary disease (COPD). Pulmonary embolism resembles COPD exacerbation so closely that these 2 entities are often impossible to distinguish clinically (3). The reported incidence of PE in studies done postmortem of patients with COPD ranges from 28% to 51% (4, 5). Pulmonary embolism is known to increase the rate of death from COPD at 1 year (6), but the clinical probability of PE and the value of noninvasive tests to rule out the diagnosis in patients with COPD have not yet been clearly assessed. To date, 2 studies have evaluated PE in patients with this disorder (3, 7). In the Prospective Investigation of Pulmonary Embolism Diagnosis (PIOPED) study, Lesser and colleagues (3) examined the characteristics of 108 patients with COPD and suspected PE; 21 (19%) received diagnoses of PE by pulmonary angiography. In this population, risk factors, symptoms, and arterial blood gas values were similar in patients with and without PE. The second study (7) showed that the presence of COPD does not affect the diagnostic performance of d-dimer testing, CTA, or pulmonary angiography for PE (7). The true frequency of PE in patients with COPD in whom PE is clinically suspected ranges from 19% to 29% (3, 7-9). Thus, clinical detection of PE in these patients is particularly difficult. In this study, we prospectively evaluated PE in patients with COPD exacerbation of unknown origin and examined factors associated with the presence of PE, including the Geneva score (1). Methods Study Objectives The objectives of our study were to assess the presence of PE in patients with COPD exacerbation of unknown origin and to explore factors associated with the presence of PE, including the Geneva score. Study Group Between April 1999 and December 2002, all consecutive patients with COPD referred to the Lung Department at the 59-bed Lille University Hospital for severe exacerbation of unknown origin were assessed for PE. Chronic obstructive pulmonary disease was diagnosed and its severity was determined according to the criteria of the American Thoracic Society (10). All patients smoked or were former smokers. Patients with asthma were not included in the study. Severe exacerbation was defined as acute deterioration from a stable condition that required hospitalization. The absence of a lower respiratory tract infection (increased sputum volume and/or increased sputum purulence, fever, history of cold, and sore throat); absence of pneumothorax and iatrogenic intervention; presence of parenchymal condensation without fever and chills; or presence of a discrepancy between clinical and radiologic features and hypoxemia severity classified the exacerbation as of unknown origin. Physicians were required to discuss each case of COPD with 1 of the referring physicians. Patients requiring invasive mechanical ventilation were referred to the intensive care unit and were not included in the study. Intervention All patients were examined within 48 hours of admission to the hospital and had spiral CTA of pulmonary circulation and color Doppler and venous lower-limb ultrasonography. These are the first-line diagnostic tests for acute PE at our institution. The decision to perform additional examinations, including d-dimer determination and ventilationperfusion scanning, was left to the discretion of the attending physician. Our local ethics committee approved the study protocol, which did not require informed patient consent. Spiral CTA In 1999, spiral CTA of pulmonary circulation was performed with a Somatom Plus 4A (Siemens Medical Systems, Forchheim, Germany) using a collimation of 3 mm3 mm, a pitch of 2, and a scanning time of 0.75 second per revolution. The results were read during the clinical work-up as previously described (9, 11-13). Because the equipment at our institution was upgraded during this study, spiral CTA of pulmonary circulation between January 2000 and December 2002 was performed with a multislice spiral computed tomography (CT) scanner, using a collimation of 4 mm1 mm, a pitch of 2, and a rotation time of 0.5 second. All patients with negative results on spiral CTA had a 3-month follow-up visit after inclusion in the study to assess critical events that were potentially related to PE. A chest physician reported death, subsequent admission to the hospital, new symptoms, and use of anticoagulant medications. Ultrasonography Venous compression ultrasonography of both legs was done from the common femoral vein and including the calf vein. Lack of compressibility was considered to indicate deep venous thrombosis. Definition Patients were classified as PE positive (positive results on spiral CTA or negative results on spiral CTA and positive results on ultrasonography) or PE negative (negative results on spiral CTA and negative results on ultrasonography or negative results on spiral CTA and no recurrence of PE at follow-up 3 months later). Assessment of the Geneva Score Because the Geneva score (1) was published by the time our study ended in 2001, we evaluated this score a posteriori in our sample before reviewing the data on PE. The probability of PE was expressed as low (a score 4), intermediate (a score of 5 to 8), or high (a score 9) (Table 1) (1). Table 1. The Geneva Score and the Modified Geneva Score Statistical Analysis Statistical analysis was done by using Epi Info software, version 3.3.2 (Centers for Disease Control and Prevention, Atlanta, Georgia), and CIs were calculated with StatExact and Stata, version 7 (Stata Corp., College Station, Texas). We calculated risk ratios and exact CIs for the various risk factors and clinical symptoms and determined P values using the Fisher exact test. A P value less than 0.05 indicated statistical significance. Role of the Funding Source No funding was received for this study. Results Study Group A total of 211 consecutive patients with COPD were referred for severe exacerbation of unknown origin. Fourteen patients were not included in the study because the results of the spiral CTA and ultrasonography were inconclusive (8 patients) or because of iodine intolerance (6 patients). Thus, the study group included 197 patients with COPD and severe exacerbation of unknown origin. There were 165 men and 32 women, and their mean age was 60.5 years (SD, 12.1). A total of 136 patients (69%) were referred from the emergency department, and 61 (31%) were inpatients who developed severe exacerbation while hospitalized. Arterial blood gas values on room air were 61.9 mm Hg (SD, 10.9) for Pao 2 and 42 mm Hg (SD, 9) for Paco 2. The mean number of risk factors for PE per patient was 0.87 (SD, 0.7). In 160 of the 197 study patients, results of a pulmonary function test performed within 3 months of the severe exacerbation were available. The mean FEV1 was 1.56 L (SD, 0.6), 52% (SD, 19%) of the predicted value. The mean FEV1vital capacity ratio was 56.4% (SD, 14.8%). The severity of respiratory disease was assessed according to the criteria of the American Thoracic Society (10): grade I, FEV1 greater than 50% of the predicted value (66 patients [41%]); grade II, FEV1 between 35% and 50% of predicted (67 patients [42%]); and grade III, FEV1 less than 35% of predicted (27 patients [17%]). Forty-nine (25%) patients were receiving long-term oxygen therapy. Pulmonary Embolism All patients had spiral CTA (37 patients had a single-slice CT scan, and 160 had a multislice CT scan), and 180 had venous ultrasonography (Table 2). None of the 197 study patients were thought to have clinical recurrence of PE during the 3 months of follow-up. Forty-three patients had positive results on CT. Twenty-five patients had deep venous thrombosis on ultrasonography; of these patients, 6 had negative results on spiral CTA. Nineteen (44%) of the 43 patients with positive results on spiral CTA also had positive results on ultrasonography. One hundred forty-eight patients did not have PE, on the basis of negative results on CT and ultrasonography and negative findings at 3-month follow-up. Thus, the prevalence of PE in our study group was 49 of 197 patients (25% [95% CI, 19% to 32%]). Table 2. Results of Spiral Computed Tomography Angiography in Patients Initially Referred for Suspected Acute Pulmonary Embolism Clinical Characteristics according to the Presence or Absence of Pulmonary Embolism The 49 patients with COPD who had PE did not differ statistically significantly from the 148 patients with COPD who did not have PE in terms of referral location (data not shown). We performed a bivariate analysis of baseline characteristics (Table 3) and clinical characteristics at admission (Table 4) that were potentially associated with PE. Clinical symptoms, such as change in dyspnea, pleuritic pain, hemoptysis, tachycardia (pulse rate >100 beats/m

Peroxisome proliferator-activated receptor γ activators affect the maturation of human monocyte-derived dendritic cells

Peroxisome proliferator‐activated receptor γ  (PPARγ ), a member of the nuclear receptor superfamily, has recently been described as a modulator of macrophage functions and as an inhibitor of T cell proliferation. Here, we investigated the role of PPARγ  in dendritic cells (DC), the most potent antigen‐presenting cells. We showed that PPARγ  is highly expressed in immature human monocyte‐derived DC (MDDC) and that it may affect the immunostimulatory function of MDDC stimulated with lipopolysaccharide (LPS) or via CD40 ligand (CD40L). We found that the synthetic PPARγ  agonist rosiglitazone (as well as pioglitazone and troglitazone) significantly increases on LPS‐ and CD40L‐activated MDDC, the surface expression of CD36 (by 184% and 104%, respectively) and CD86 (by 54% and 48%), whereas it reduces the synthesis of CD80 (by 42% and 42%). Moreover, activation of PPARγ  resulted in a dramatic decreased secretion of the Th1‐promoting factor IL‐12 in LPS‐ and CD40L‐stimulated cells (by 47% and 62%), while the production of IL‐1β , TNF‐α , IL‐6 and IL‐10 was unaffected. Finally, PPARγ  ligands down‐modulate the synthesis of IFN‐γ ‐inducible protein‐10 (recently termed as CXCL10) and RANTES (CCL5), both chemokines involved in the recruitment of Th1 lymphocytes (by 49% and 30%), but not the levels of the Th2 cell‐attracting chemokines,macrophage‐derived chemokine (CCL22) and thymus and activation regulated chemokine (CCL17), in mature MDDC. Taken together, our data suggest that activation of PPARγ  in human DC may have an impact in the orientation of primary and secondary immune responses by favoring type 2 responses.

From parasites to allergy: a second receptor for IgE

Human allergic responses are triggered when mast cells bind IgE antibodies. IgE production is also a regular feature of helminth infection and parasites can be killed by inflammatory cells such as macrophages, eosinophils and platelets sensitized by reaginic antibody. The IgE receptor they use has been identified only recently. Here André Capron and his colleagues discuss how it differs from the IgE receptor present on mast cells.

Endocan is a novel chondroitin sulfate/dermatan sulfate proteoglycan that promotes hepatocyte growth factor/scatter factor mitogenic activity.

Proteoglycans that modulate the activities of growth factors, chemokines, and coagulation factors regulate in turn the vascular endothelium with respect to processes such as inflammation, hemostasis, and angiogenesis. Endothelial cell-specific molecule-1 is mainly expressed by endothelial cells and regulated by pro-inflammatory cytokines (Lassalle, P., Molet, S., Janin, A., Heyden, J. V., Tavernier, J., Fiers, W., Devos, R., and Tonnel, A. B. (1996) J. Biol. Chem. 271, 20458–20464). We demonstrate that this molecule is secreted as a soluble dermatan sulfate (DS) proteoglycan. This proteoglycan represents the major form either secreted by cell lines or circulating in the human bloodstream. Because this proteoglycan is specifically secreted by endothelial cells, we propose to name it endocan. The glycosaminoglycan component of endocan consists of a single DS chain covalently attached to serine 137. Endocan dose-dependently increased the hepatocyte growth factor/scatter factor (HGF/SF)-mediated proliferation of human embryonic kidney cells, whereas the nonglycanated form of endocan did not. Moreover, DS chains purified from endocan mimicked the endocan-mediated increase of cell proliferation in the presence of HGF/SF. Overall, our results demonstrate that endocan is a novel soluble dermatan sulfate proteoglycan produced by endothelial cells. Endocan regulates HGF/SF-mediated mitogenic activity and may support the function of HGF/SF not only in embryogenesis and tissue repair after injury but also in tumor progression.

Human Endothelial-Cell Specific Molecule-1 Binds Directly to the Integrin CD11a/CD18 (LFA-1) and Blocks Binding to Intercellular Adhesion Molecule-1

ICAMs are ligands for LFA-1, a major integrin of mononuclear cells involved in the immune and inflammatory processes. We previously showed that endothelial cell specific molecule-1 (ESM-1) is a proteoglycan secreted by endothelial cells under the control of inflammatory cytokines. Here, we demonstrate that ESM-1 binds directly to LFA-1 onto the cell surface of human blood lymphocytes, monocytes, and Jurkat cells. The binding of ESM-1 was equally dependent on Ca2+, Mg2+, or Mn2+ divalent ions, which are specific, saturable, and sensitive to temperature. An anti-CD11a mAb or PMA induced a transient increase in binding, peaking 5 min after activation. Direct binding of ESM-1 to LFA-1 integrin was demonstrated by specific coimmunoprecipitation by CD11a and CD18 mAbs. A cell-free system using a Biacore biosensor confirmed that ESM-1 and LFA-1 dynamically interacted in real time with high affinity (Kd = 18.7 nM). ESM-1 consistently inhibited the specific binding of soluble ICAM-1 to Jurkat cells in a dose-dependent manner. These results suggest that ESM-1 and ICAM-1 interact with LFA-1 on binding sites very close to but distinct from the I domain of CD11a. Through this mechanism, ESM-1 could be implicated in the regulation of the LFA-1/ICAM-1 pathway and may therefore influence both the recruitment of circulating lymphocytes to inflammatory sites and LFA-1-dependent leukocyte adhesion and activation.

Release of granule proteins by eosinophils from allergic and nonallergic patients with eosinophilia on immunoglobulin-dependent activation

The release of eosinophil peroxidase (EPO) and eosinophil cationic protein (ECP) was evaluated after incubation of eosinophils (EOSs) from allergic subjects with the specific allergen or with anti-IgE monoclonal antibodies (MAbs). High levels of EPO could be released after addition of the specific allergen (and not unrelated ones) or anti-IgE MAb. Moreover, EPO release with the two stimuli was significantly correlated both in allergic and in nonallergic patients. In the same supernatants, another granule protein, ECP, could not be detected, suggesting a lack of correlation between EPO and ECP release after IgE-dependent stimulation. However, when EOSs with surface-IgA antibodies were incubated with anti-IgA MAb, both EPO and ECP were released. In contrast, incubation of EOSs with anti-IgG MAb induced mainly the release of ECP and not EPO. These results indicate that pharmacologically active mediators can be released by EOSs from allergic and nonallergic patients on immunoglobulin-dependent activation. The results also confirm the hypothesis of a selective release of the various granule proteins and raise the question of transduction signals delivered by the three Fc receptors (Fc epsilon R, FC alpha R, and FC gamma R) present on human EOSs.

Characterization of the Secreted Form of Endothelial-Cell-Specific Molecule 1 by Specific Monoclonal Antibodies

Endothelial-cell-specific molecule 1 (ESM-1) is a recently identified endothelial cell molecule. As ESM-1 mRNA is preferentially expressed in human lung and kidney tissues, and as ESM-1 mRNA expression is regulated by inflammatory cytokines, ESM-1 is thought to play a role in the vascular contribution to organ-specific inflammation. In order to define its behavior, mouse anti-ESM-1 monoclonal antibodies were developed, and three distinct epitopes were mapped, which allowed development of a specific ELISA assay, immunohistological staining and immunoblot analysis. Here, we demonstrate that ESM-1 is present in cell lysates of human endothelial cells (human umbilical vein endothelial cells) with an apparent molecular weight of 20 kD. In contrast, the secreted form of ESM-1 is shifted to an apparent molecular weight of 50 kD, indicating that the secreted form of ESM-1 is posttranslationally modified. By ELISA, we show that the secretion of ESM-1 is significantly enhanced in the presence of TNFα. In contrast, the spontaneous as well as TNFα-induced secretion of ESM-1 is strongly inhibited by IFNγ. Moreover, ESM-1 was detected in the serum of healthy subjects at an average concentration of 1.08 ng/ml, and we demonstrated that the serum level of ESM-1 is dramatically increased in patients presenting a septic shock. Analysis of ESM-1 expression in normal human tissues by immunohistochemistry showed that ESM-1 is localized in the vascular network, but also in the bronchial and renal epithelia. Our results demonstrate that ESM-1 is mainly expressed in the vascular endothelium both in vitro and in vivo, but also by different epithelia. ESM-1 may represent a new marker of endothelial cell activation, and may have a functional role in endothelium-dependent pathological disorders.

Prostaglandin D2 Affects the Maturation of Human Monocyte-Derived Dendritic Cells: Consequence on the Polarization of Naive Th Cells

Among the factors produced at inflammatory sites and those capable of modulating dendritic cell (DC) functions, PGD2 may be important in the outcome of immune responses. The biological roles for PGD2 are in part effected through two plasma membrane G protein-coupled receptors: the D prostanoid (DP) receptor and the chemoattractant receptor-homologous molecule expressed on Th2 lymphocytes (CRTH2). In this report, we studied the effects of PGD2 and of its major physiological metabolite, 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2), on the functions of human monocyte-derived DC. First, we show that PGD2 exerts in vitro chemotactic effects on monocytes via CRTH2 activation while it inhibits the chemokine-driven migration of monocyte-derived DC through DP. We also report that PGD2 and 15d-PGJ2 alter the LPS- and allergen-induced DC maturation and enhance the CD80/CD86 ratio on mature DC in a DP- and CRTH2-independent manner. Moreover, PGD2 and 15d-PGJ2 strongly reduce the secretion of the Th1 promoting cytokine IL-12 and affect the synthesis of chemokines involved in Th1 cell chemotaxis, particularly CXCL10. Inhibition of cytokine/chemokine secretion implicates at least in part DP, but not CRTH2. The effects exerted by PGD2 are associated with the phosphorylation of CREB, but do not parallel with the deactivation of the NF-κB and mitogen-activated protein kinase pathways. In contrast, 15d-PGJ2 seems to target other cellular proteins. Finally, in a model of Th CD45RA+ differentiation induced by allergen- and superantigen-pulsed DC, PGD2 impacts on the orientation of the immune response by favoring a Th2 response.