Abstract
Our initial observation that type I collagen activates polymorphonuclear leukocytes (PMN) prompted the testing of the activating potential of type IV collagen. It was noted, however, that type IV collagen isolated from bovine lens capsule did not activate PMN but rather prevented their stimulation by N-formylmethionyl-leucyl-phenylalanine, phorbol myristate acetate, or type I collagen. This observation led to the present study, which demonstrates that the inhibitory effect of lens capsule type IV collagen resides in the noncollagenous (NC1) domain of the alpha 3 chain and specifically in the region comprising residues 185-203 of the NC1 domain of both the human and bovine molecules. Synthetic peptides from the same region of the NC1 domains of the alpha 1, alpha 2, alpha 4, and alpha 5 chains did not possess the inhibitory effect seen with the alpha 3 chain. The sequence S-N-S (residues 189-191) is unique to the peptide of the alpha 3 chain, and substitution of either serine with alanine abolishes the inhibition. Type IV collagen isolated from the mouse Engelbreth-Holm-Swarm (EHS) tumor, a molecule that lacks the alpha 3 chain, did not prevent PMN activation but instead stimulated the secretion of elastase and type IV collagenase. Incubation of PMN with intact lens capsule type IV collagen or a peptide comprising residues 185-203 of the alpha 3 (IV) chain resulted in a 3-fold increase of intracellular cAMP, whereas, Ca2+ levels remained unchanged. Incubating PMN with forskolin or with dibutyryl-cAMP resulted in the inhibition of O2- production and degranulation by PMN, thus mimicking the effects of type IV collagen and the alpha 3 (IV) 185-203 peptide. The data suggest that type IV collagen, through its alpha 3 chain, down-regulates PMN activation and thus decreases the potential for damage as these cells traverse the capillary wall. Our in vitro experiments suggest that the higher the content of the alpha 3 (IV) chain is in a basement membrane, the wider would be its capacity for self-protection.
Highlights
Our initial observation that type I collagen activates to-end interaction involving the noncollagenous (NC1)‘carpolymorphonuclearleukocytes (PMN) prompted the boxyl termini
In view of the potential for damage to surrounding tissuebsy the productsof PMN activation and the close contact of PMN with the basement memlacks the a3 chain, did not prevent PMN activation but brane, as thesecells extravasate through thecapillary wall, it instead stimulated the secretion of elastase and type IV becomes important to understand the mechanisms that regucollagenase
The data suggest that type IV collagen, through its a3 chain, down-regulates PMN activation and decreases the potential for damage as these cells traverse the capillary wall
Summary
Collagen healthy subjects and PMN were isolated accordtinoga previously published method( l l ) , by a single-step centrifugation procedure througha. Measurement of Superoxide Anion (0;)"o; release was measured ligand solution containing either pg of ALC type IV collagen, 20 pg from the superoxide dismutase-inhibitable reductioonf ferricytochrome of EHS typeIV collagen, or 20 pg of the bovine peptide a3(IV) 185-203 c according to the method of English et al [22] as described by Mon- and the mixture incubated for 0.5, 1, 2, 5, 15, 30, 45, and 60 min at boisse et al (IO).The increase in absorbanacte550 nm was takeans the 37 "C. A PMN suspension (1.2 ml containing 6 x lo cells) in Dulbecco's solution was incubated for 30 min a t 37 "C with type IV collagen or its supernatants were neutralized with 9 M NaOH and acetylated before analysis.Cytoplasmic CAMP wasthenmeasuredaccordingtothe method of Cailla etal.
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