Skin reactivity and local cell recruitment in human atopic and nonatopic subjects by CCL2/MCP-1 and CCL3/MIP-1alpha.

BACKGROUND: Monocyte chemotactic protein (MCP-1/CCL2), the ligand for CCR2 and CCR5, and macrophage inflammatory protein-1alpha (MIP-1alpha/CCL3), the ligand for CCR1 and CCR5, are potent chemo-attractants in vitro and produce lesions in experimental animals, which resemble immediate and delayed-type hypersensitivity (DTH) reactions. CCL3 induces mononuclear cell and granulocyte infiltration in human atopic and nonatopic skin. Whether CCL2 (MCP-1) has comparable activity in man is uncertain as is the capacity of both the chemokines to elicit immediate- and DTH-like reactions in humans. METHODS: Inflammatory cells were counted by immunohistochemistry in 24 and 48-h skin biopsies from atopics and nonatopics after intradermal injection of CCL2 and CCL3. Immediate (15 min) wheals-and-flares and delayed (24 and 48 h) indurations were also recorded. RESULTS: Both chemokines induced immediate- (15 min) and delayed (24 and 48 h) reactions, which were associated with significant infiltrations of CD68+ macrophages, CD3+, CD4+ (but not CD8+) T cells, neutrophils, and eosinophils in biopsies from injection sites. CCL2, but not CCL3, also induced infiltration of basophils. Neither chemokine produced significant changes in the numbers of tryptase+ cutaneous mast cells. There were no differences in the pattern of skin reactivity or the numbers of infiltrating leukocytes in response to CCL2 and CCL3 between atopic and nonatopic subjects. In general, maximal infiltration of inflammatory cells was observed at the 24-h, rather than the 48-h, time point. CONCLUSIONS: CCL2 and CCL3 induce both immediate and delayed skin reactions in atopics and nonatopics, and evoke a similar profile of local T cell/macrophage and granulocyte recruitment which, in general, confirm previous in vitro findings and in vivo experimental animal data.

Airway expression of calcitonin gene-related peptide in T-cell peptide-induced late asthmatic reactions in atopics.

BACKGROUND: The mechanisms of late asthmatic reactions provoked in atopic asthmatics by allergen-derived T-cell peptide epitopes remain unclear. Previous studies showed no changes in airway eosinophils or mast cell products after peptide challenge. In the present study our aim was to measure calcitonin gene-related peptide (CGRP), neurokinin (NK)-A, and substance P (SP) in bronchoalveolar lavage fluid and bronchial biopsies (BB) after inhalation of allergen-derived T-cell peptide epitopes since these neuropeptides (NP) had not previously been evaluated in this chronic asthma model. METHODS: Bronchoscopy, with BB and bronchoalveolar lavage (BAL), was performed in 24 cat-allergic subjects 6 h after inhalation of Fel d 1-derived peptides. Neuropeptides were measured in BAL by enzyme-linked immunosorbent assay and CGRP expression in the airways was assessed by immunohistochemistry and confocal microscopy. RESULTS: Twelve subjects (termed 'responders') developed isolated late reactions. Calcitonin gene-related peptide, but not NK-A or SP, was significantly elevated in BAL in responders only. Biopsy studies showed that in virtually all responders peptide challenge induced marked increases in CGRP immunoreactivity in bronchial epithelial cells, infiltrating submucosal cells and in association with airway smooth muscle. Double immunostaining indicated that CGRP colocalized predominantly to CD3+/CD4+ and CD68+ submucosal inflammatory cells. CONCLUSION: Calcitonin gene-related peptide, a potent vasodilator, is markedly up-regulated in the airways of atopic asthmatics during late-phase reactions provoked by inhalation of allergen-derived T-cell peptides.

Bacillus subtilis levansucrase: amino acid substitutions at one site affect secretion efficiency and refolding kinetics mediated by metals.

Studies of the equilibrium between native and denatured forms of wild-type levansucrase showed that the denatured form was predominant at 37 degrees C and pH 7 in the absence of free metal. The shift to the native form was promoted by metal ions such as Fe3+ or Ca2+. This metal-dependent refolding process was not observed in levansucrase variants bearing the amino acid substitution Gly-366----Asp or Gly-366----Val. These variants were only slightly secreted by Bacillus subtilis although their signal sequences were normally cleaved and their exocellular forms stable. In contrast, the Gly-366----Ser variant was secreted at near-normal levels and shared a part of the in vitro refolding properties of the wild-type protein. These differential properties might be related to the ability of the altered region to form a beta-turn structure. We discuss the possible role of metal ions in the coupling of protein folding and secretion.

Secretion of Bacillus subtilis levansucrase. Fe(III) could act as a cofactor in an efficient coupling of the folding and translocation processes.

The refolding of levansucrase denatured by urea was studied as a possible model for the second step of the secretion pathway of this protein. The folding-unfolding transition was monitored by measuring intrinsic fluorescence and resistance to proteolysis. Both methods provided the same estimation for the unfolding free energy of levansucrase, delta GD, which was 30.1 +/- 1.7 kJ.mol-1 (7.2 +/- 0.4 kcal.mol-1) at pH 7 in 0.1 M-potassium phosphate buffer. The rate of refolding was greatly enhanced by Fe3+, whereas the Fe3+ chelator EDTA prevented correct refolding. Fe3+ allowed the protein to reach its folded form in medium in which the dielectric constant had been lowered by ethanol. The efficiency in vivo of the export of levansucrase bearing an amino acid modification which blocks the second step of the translocation pathway was greatly increased by high concentrations of Fe3+ in the culture medium. Assuming that the protein folding governs the second step of the secretion process of levansucrase, we discuss from an irreversible thermodynamic point of view the possible role of Fe3+ in the efficient coupling of the two events.

Levansucrase of Bacillus subtilis: effects on the secretion process of single amino acid substitutions in the mature part of the protein.

Substitutions of an aspartate or an arginine residue for the glycine residue at position 366 of the mature part of Bacillus subtilis levansucrase were obtained by mutagenesis. Quantitative estimation from immunoblot analysis showed that the two transient membrane forms of the modified proteins were present in the membrane at the same level as that of the wild-type protein. The proteolytic processing, which was previously shown to be the first step of the levansucrase secretion process, was not affected in these modified proteins. Results from pulse-chase experiments showed that the half-times for secretion of the modified levansucrases into the culture medium were nearly the same as that of the wild-type protein, but the amount of the modified proteins secreted was significantly reduced. Purified samples of the modified enzymes were subtilisin insensitive and possessed enzyme activities very similar to those of the wild-type enzyme. The results suggest that the 366 site probably belongs to a functional domain of the protein which could play an important role in the second step of the levansucrase secretion process.

Secretion of Bacillus subtilis levansucrase: a possible two-step mechanism.

The rate of exocellular levansucrase synthesis in an overproducing (sacUh) strain of Bacillus subtilis was shown to be directly proportional to the amount of two different transient forms of this enzyme located within the membrane fraction of the cells. The apparent Mr of the larger membrane form was 53,000, and that of the smaller form 50,000; the half-life time of each form was estimated in vivo to be 4-6 s and 32-42 s, respectively. Ethanol treatment of the cells lead to the accumulation of the 53,000-Mr form which may represent 1.5% of total membrane proteins. This latter form, partially purified, was transformed in vitro into the 50,000-Mr form by the action of the Escherichia coli leader peptidase. These enzyme forms were quite different from the exocellular levansucrase since they showed a weak affinity for hydroxyapatite and needed complexed iron to display enzyme activity. Assuming the membrane forms were precursors of exocellular levansucrase, we propose a two-step mechanism for the secretion process of levansucrase. The number of exoprotein synthesis/secretion sites in a B. subtilis cell is estimated to 2.5 X 10(4).