Joachim Lundahl

Tissue Inhibitor of Metalloproteinase 1 Activates Normal Human Granulocytes, Protects Them from Apoptosis, and Blocks Their Transmigration during Inflammation

ABSTRACT Urinary levels of tissue inhibitor of metalloproteinase 1 (TIMP-1) higher than those of matrix metalloproteinase 9 (MMP-9) during acute pyelonephritis have previously been associated with a higher degree of acute inflammation and of postinfective renal scarring. The aim of the present study was to evaluate possible mechanisms by which TIMP-1 could affect the scarring process already during the acute phase of inflammation. The growth of Escherichia coli, bactericidal activity of fresh human blood, and respiratory burst, spontaneous apoptosis, and trans-basement membrane migration of normal human granulocytes were studied in vitro in the presence of different concentrations of recombinant human TIMP-1. To imitate the “normal” environment during inflammation in the kidney, granulocytes were also incubated with a conditioned medium from E. coli-stimulated renal epithelial cells. In order to compare our data with the in vivo situation, blood and urinary leukocyte levels were analyzed for 40 children with acute pyelonephritis, together with urinary MMP-9 and TIMP-1 levels. TIMP-1 at a concentration of 500 ng/ml increased the bactericidal activity of blood, increased the respiratory burst of granulocytes, decreased phosphatidylserine exposure and caspase 3 activity, which are features of spontaneous apoptosis, and inhibited granulocyte transmigration. Moreover, in the patients with pyelonephritis, MMP-9/TIMP-1 ratios in urine correlated with the degree of leukocyte transmigration. Thus, our data suggest that TIMP-1 specifically blocks the transmigration of granulocytes into urine. Entrapped and activated granulocytes, protected from apoptosis, might excessively destroy renal parenchyma and thus contribute to the pathogenesis of renal scarring following acute pyelonephritis.

Simplified quantitation of myeloid dendritic cells in peripheral blood using flow cytometry

BACKGROUNDnRecognition of the importance of dendritic cells (DC) in the initiation of T-cell-dependent immune responses has led to increasing interest in methods for the identification of DC within the circulation. We sought to develop a flow cytometric method that would allow the reliable enumeration of absolute myeloid DC counts in minimally manipulated blood samples.nnnMETHODSnMyeloid DC were identified by three-color staining of whole blood leukocytes as a discrete population of mononuclear cells expressing high levels of HLA-DR and CD33, yet having little or no expression of CD14 and CD16. This method was analyzed for reproducibility and variation in blood DC number during typical clinical day hours and after exercise. The new method was compared to an established commercial kit method.nnnRESULTSnFACS sorting of the CD33(+) DC showed that they morphologically resembled immature DC, and developed cytoplasmic projections typical of mature DC following overnight culture in granulocyte macrophage-colony stimulating factor (GM-CSF). Within peripheral blood, these DC were found at a mean concentration of 17. 4 +/- 5.4 x 10(6) per liter, corresponding to 0.93 +/- 0.27% of mononuclear cells. Comparison of duplicate samples stained and analyzed in parallel showed that the intrasample variability was very low, with an intraclass correlation coefficient of 0.95. The frequency of CD33(+) myeloid DC and their light scatter characteristics were similar to that of CD11c(+) myeloid cells. Four-color FACS analysis revealed complete identity of CD11c(hi), HLA-DR(+) DC with CD33(+), HLA-DR(+) DC. Only rare CD33(+) DC coexpressed CD123 and HLA-DR. Numbers of blood myeloid DC, identified by CD33 staining, showed no significant variation during standard laboratory hours. However, their numbers rose significantly during vigorous exercise, in parallel to other blood cells.nnnCONCLUSIONSnThe method described herein is rapid, reproducible, requires only small volumes of blood, can be readily used by a clinical immunology laboratory, and requires fewer antibodies than a currently available commercial method.

Difference in the Blood Monocyte Phenotype Between Uremic Patients and Healthy Controls: Its Relation to Monocyte Differentiation into Macrophages in the Peritoneal Cavity

The phenotypic alterations between blood monocytes from 11 patients with end-stage renal disease, who had been on peritoneal dialysis for less than one week, and blood monocytes from 10 healthy controls, were analyzed. In addition, peritoneal macrophages in the dialysate effluent were enclosed. Analysis of functional receptor density was performed using immunostaining and flow cytometry. The phenotypic characterization was selected to represent various biological functions such as adhesion, phagocytosis (CD11b/CD18, CD11c/CD18, CD16), antigen-presentation (HLA-DR, ICAM-1), differentiation (transferrin receptor, CD71), receptor for LPS (CD14) and initiation of the coagulation cascade (Tissue factor, CD142). The proportion of CD16-positive blood monocytes and the quantitative level of ICAM-1 were higher in the patient group, compared to healthy controls. A significant increase in the quantitative level of CD11b/CD18, CD11c/CD18, HLA-DR and ICAM-1, transferrin receptor, CD 14 and CD 16, was found on peritoneal macrophages, compared to monocytes, harvested both from the corresponding patients, as well as from healthy donors. In contrast, we did not find any significant differences in the expression of tissue factor between monocytes and peritoneal macrophages. In conclusion, phenotypic differences exist between monocyte populations in the blood circulation of CAPD patients, and healthy individuals. We also show that transmigration of monocytes into the peritoneal cavity implies a selective up-regulation of functional receptors, preferentially related to adhesion, and antigen-presentation in a steady-state situation in non-infected CAPD patients.

Eotaxin Increases the Expression of CD11b/CD18 and Adhesion Properties in IL5, But Not fMLP-Prestimulated Human Peripheral Blood Eosinophils

A selective recruitment of eosinophils to sites of inflammation is claimed to be controlled by regulation of cytokines, chemokines and adhesion molecules. In animal models, eotaxin has been suggested to be a potent chemokine since it in cooperation with interleukin-5 induce selective chemotaxis and infiltration of eosinophils to lung tissue after an allergen provocation. We have investigated the in vitro effect of eotaxin on human peripheral blood eosinophils with respect to CD11b/CD18 expression and adhesion properties to the matrix protein fibronectin. We did not find any effect of eotaxin per se on CD11b/CD18 expression, neither on eosinophils from healthy subjects nor from patients with asymptomatic pollen related asthma. However, eotaxin significantly upregulated the quantitative level of CD11b/CD18 and increased the adhesion to fibronectin in eosinophils from healthy subjects preincubated in vitro with interleukin-5, but not in eosinophils preincubated with fMLP. Moreover, eosinophils harvested 24 hours after an in vivo allergen inhalation provocation in asthmatics, upregulated CD11b/CD18 after in vitro incubation with eotaxin alone.

Gene expression analysis using long-term preserved formalin-fixed and paraffin-embedded tissue of non-small cell lung cancer.

This study was performed to evaluate RNA extraction and gene expression analysis of non-small cell lung cancer (NSCLC) using formalin-fixed and paraffin-embedded (FFPE) specimens stored for more than 20 years by quantitative PCR (qPCR) and DNA microarrays. Long-term preserved FFPE materials enable large retrospective studies correlating molecular features with therapeutic response and clinical outcome. qPCR was used to evaluate RNA extraction methods and to compare DNA microarray gene expression profiles of FFPE and fresh frozen (FF) tissue. The Ambion RecoverAll kit appeared to be suited for RNA extraction of long-term preserved FFPE tissues. Microarray analysis using the Affymetrix platform displayed a high degree of correlation for endogenous control genes comparing FF and FFPE tissues and identified known NSCLC signature genes in both specimens. We conclude that high quality gene expression signatures can be recognized using the Affymetrix gene expression platform on FFPE tissue stored for more than 20 years. However, a general interpretation must be done with caution as different FFPE procedures have varying effects on RNA quality.

Reduced expression of hemopoietic cytokine receptors on cord blood progenitor cells in neonates at risk for atopy.

BACKGROUNDnRecent findings point to an association between allergic asthma in adults and increased responsiveness of myeloid progenitor cells to certain hemopoietic growth factors. However, it is not clear at what age these changes in progenitor cells first become manifest, although increasing evidence suggests that the allergic phenotype may begin to emerge in very early life.nnnOBJECTIVEnWe sought to compare expression of hemopoietic cytokine receptors on CD34(+) progenitor cells in cord blood from normal infants (low risk for subsequent atopy) and infants with at least one atopic first degree relative (at risk for subsequent atopy).nnnMETHODSnCord blood was obtained from 21 neonates. Nonadherent mononuclear cells were stained with mAbs directed against CD45, CD34, and the alpha-chains of the GM-CSF, IL-3, and IL-5 receptors and analyzed by flow cytometry.nnnRESULTSnNo differences in absolute CD34(+) numbers were observed between the 2 groups. However, expression of GM-CSF receptor on CD34(+) cells was reduced in the at-risk compared with the low- risk group (P =.021), although no significant differences were noted between the 2 groups with respect to IL-3 and IL-5 receptor expression.nnnCONCLUSIONnThe functional sequelae of reduced GM-CSF receptor expression on CD34(+) cells remain to be determined. Nonetheless, these findings show an association between genetic risk for atopy and changes in the expression of hemopoietic cytokine receptors on cord blood progenitor cells and support the notion that the allergic phenotype may begin to evolve in the perinatal period.

Staphylococcus epidermidis Isolated From Newborn Infants Express Pilus-Like Structures and Are Inhibited by the Cathelicidin-Derived Antimicrobial Peptide LL37

Coagulase-negative staphylococci and its subtype Staphylococcus epidermidis are major indigenous Gram-positive inhabitants of the human skin. Colonization occurs in direct connection with birth and terrestrial adaptation. This study focuses on factors that may influence skin colonization of the newborn infant that relates to the immune status of both the bacteria and the host. Skin is an effective barrier against bacteria, and this function is partly mediated by the presence of antimicrobial peptides including human cathelicidin peptide LL37. Gram-positive bacteria have been described to have adhesive pili on their surface that mediates specific attachment to the host. Here, we identify, by negative staining transmission electron microscopy (EM), two different types of pilus-like structures commonly expressed on S. epidermidis isolated from newborn infants. We also show that the cathelicidin antimicrobial peptide LL37, constitutively expressed in the skin barrier of the newborn, significantly inhibited growth of S. epidermidis indicating its importance for the ecological stability of the skin microbiota. Further studies are required to elucidate molecular mechanisms of host-microbe interactions, both for the maintenance of a mutually beneficial homeostatic relationship and for the protection of self when it results in overt disease.

Effect of nasal steroid treatment on airway inflammation determined by exhaled nitric oxide in allergic schoolchildren with perennial rhinitis and asthma

Rhinitis is common in asthmatic schoolchildren who are allergic to animal dander and constantly and indirectly exposed to these allergens in their everyday environment. As a patho‐physiological linkage between nasal and bronchial inflammation has been proposed to exist, the primary objective of this study was to determine whether nasal administration of mometasone furoate (MSNF) can reduce bronchial inflammation, as reflected in the level of exhaled nitric oxide (FENO) in asthmatic schoolchildren with dander allergy and mild‐to‐moderate rhinitis. Forty such children were assigned randomly to be treated for 4u2003wk with MSNF or placebo, employing a double‐blind procedure. FENO was the primary end‐point measured and secondary end‐points were nasal levels of NO, the concentration of eosinophilic cationic protein (ECP) in nasal lavage, the relative numbers of eosinophils in blood, forced expiratory volume in 1u2003s (FEV1), peak expiratory flow (PEF) and scoring of symptoms. There was no significant difference in the FENO values of the treated and control groups at any time‐point, whereas the nasal level of ECP was lower in the treated group compared with placebo (pu2003=u20030.05) on both days 7 and 28, and compared with baseline for the treated group (pu2003=u20030.06 on day 7, pu2003=u20030.02 on day 28). Furthermore, the mean blood eosinophil count decreased in the treated group, which also demonstrated lower scores for nasal symptoms compared with placebo, but neither of these differences were statistically significant. FEV1, PEF and nasal levels of NO remained unchanged in both groups. Four weeks of nasal treatment with MSNF had no effect on bronchial inflammation, as reflected by exhaled NO, whereas signs of nasal and systemic eosinophil activation were reduced. Thus, nasal administration of a steroid as a strategy to reduce asthmatic inflammation remains questionable in mild‐to‐moderately severe cases of perennial rhinitis and asthma.

Down-regulated IL-5 receptor expression on peripheral blood eosinophils from budesonide-treated children with asthma

Background:u2002The expression and function of cytokine receptors on peripheral blood eosinophils (PBE) from healthy and asthmatic children are poorly characterized.

Selective upregulation of a functional β7 integrin on differentiating eosinophils

Background:u2002The sequence of adhesion‐molecule expression during eosinophil differentiation remains unclear.