Elastase, a marker for neutrophils in skin infiltrates

Abstract

The enzyme elastase (EC 3.4.21.37) has proved to be a convenient and extremely sensitive marker for the quantification of neutrophils in cutaneous infiltrates. Fluorometric assay using the synthetic substrate MeOSuc-Ala-Ala-Pro-Val-N-methylcoumarin permitted the measurement of this enzyme in as few as five cells and was linear up to about 1000 cells per sample. The mean activity of lysates of human blood-derived neutrophils was 0.57 +/- 0.08 pmol of 7-amino-4-methyl-coumarin released per hour per neutrophil. Extracts of normal human skin contained no measurable elastase activity but resulted in a slight inhibition of the neutrophil enzyme (mean 12%). Application to the in vivo situation has been demonstrated by the use of leukotriene B4 as chemotactic agent. A reproducible neutrophil infiltrate was found at a dose of 2 ng, well below the threshold for the appearance of microabscesses.

Inhibitory Effect of an Extract of Sanguisorba officinalis L. on Ultraviolet-B-Induced Photodamage of Rat Skin.

“…18) Neutrophil elastase has been reported to degrade elastic fibers [5][6][7][8] more markedly than does fibroblast-derived elastase. 7) Therefore, it is possible that activation of neutrophil elastase by UVB irradiation damages the 3-dimensional structure of dermal elastic fibers.…”

“…Elastin has been reported to be degraded mainly by serine protease and by metalloprotease. [5][6][7][8][9][10][11][12] When elastase activity was measured in the rat skin after chronic UVB irradiation, phosphoramidon-sensitive metalloprotease activity was increased. 14,15) Therefore, an inhibitor of fibroblast elastase (a member of the metalloprotease family 12,16) ) was synthesized and was applied topically after each UVB irradiation.…”

“…Subsequently, to evaluate the cause of elastic fiber curling, we noted that elastase is activated by UVB irradiation. Elastic fibers have been reported to be digested by serine proteinase in infiltrating cells that migrate after UVB irradiation, [5][6][7][8] by MMP-12 derived from macrophages, 9,10) and by elastase produced by fibroblasts.11,12) Chatterjee et al reported an increase in elastase activity after chronic UVB irradiation at a suberythemal dose in the skin of hairless mice.13) Therefore, we evaluated elastase activity in rat hind limb skin after chronic UVB irradiation at a suberythemal dose and observed no increase in neutrophil elastase activity but a significant increase in phosphoramidon-sensitive metalloprotease activity.14,15) Those findings suggested that the UVB-induced wrinkle formation and the increase in skin elasticity could be inhibited, and that curling of dermal elastic fibers could be avoided, by inhibiting the increase in metalloprotease activity. We synthesized a chemical compound (N-phenetyl-Leucyl-Tryptophane; NPLT) that specifically inhibits fibroblast-derived elastase which is in the metalloprotease family, 12,16) and we applied this agent topically to rat hind limb skin immediately after UVB irradiation and 1 d later.…”

“…Subsequently, to evaluate the cause of elastic fiber curling, we noted that elastase is activated by UVB irradiation. Elastic fibers have been reported to be digested by serine proteinase in infiltrating cells that migrate after UVB irradiation, [5][6][7][8] by MMP-12 derived from macrophages, 9,10) and by elastase produced by fibroblasts. 11,12) Chatterjee et al reported an increase in elastase activity after chronic UVB irradiation at a suberythemal dose in the skin of hairless mice.…”

Effect of Leukotriene B₅ on the Accumulation of Polymorphonuclear Leukocytes in Unstimulated and Leukotriene B₄-Stimulated Human Skin

“…Twenty-four hours after the applications razor blade biop sies (3 mm diameter, 2-3 mg wet weight) were taken. Immediately after performing the biopsy elastase measurements were car ried out according to standard procedures [16,17], Briefly, after homogenizing in cetrimide buffer elastase activity was mea sured in the supernatant fraction from the fluorescent product released from the sub strate MeOSuc-Ala-Ala-Pro-Val-N-methylcoumarin, using a Perkin Elmer fluorometer. By the inclusion of an internal standard of elastase (equivalent to 500 PMN) the endog enous inhibition of skin was corrected.…”