Platelet factor 4 is shown to be a chemotactic protein for human polymorphonuclear leukocytes and monocytes at concentrations found in human serum and reached locally in injured tissue. The maximum chemotactic response to platelet factor 4 nearly equals that achieved with saturating concentrations ofthe chemotactic activity derived from the fifth component of human complement, C5. Cells desensitized to C5 chemotactic activity retain chemotactic responsiveness to platelet factor 4. Serum contains inhibitory capacity against the chemotactic activity associated with platelet factor 4. Our results suggest that the local release of platelet factor 4 may be an important stimulus attracting inflammatory cells to sites of blood vessel injury. Chemotaxis assays were performed in modified Boyden chambers (Ahlco, Southington, CT) by a double-filter method by using a 5-pum (monocyte) or 2-;im (neutrophil) (Nucleopore, Pleasanton, CA) filter overlaying a 0.45-pum filter (Millipore) (10). The upper compartments ofthe chambers were filled with 0.7 ml of cell suspension. The lower compartments contained 1.7 ml of medium only or medium with either platelet factor 4 or chemotactic activity derived from the fifth component of complement (C5f) that was obtained from whole serum by incubation with zymosan and e-aminocaproic acid (1.0 M) at 370C, followed by gel filtration over Sephadex G-100 (11). Twice the C5f activity necessary for 50% maximal chemotactic response (EDW) (in 25 jil) was used as a positive control in each experiment. To distinguish chemotaxis from chemokinesis, platelet factor 4 was simultaneously incubated with the cells in the upper compartment and platelet factor 4, CMf, or buffer alone in the lower compartment. In other experiments, test cells were desensitized to C5f by preincubation with C5ffor 15 min at room temperature (12). After gentle washing three times, the cells were tested promptly for chemotactic activity.For chemotaxis measurements, the chambers were incubated for 60 min (neutrophils) or 90 min, (mononuclear cells) at 37°C and then disassembled. The filters were stained with hematoxylin; after coding to conceal the identity from the reader, the filters were quantified for cell movement by recording the number of cells per high power (x400) grid migrating to the bottom of the upper filter. Five fields were averaged on each filter. Cell migration was corrected for blanks, in which the lower compartment contained only medium. Blank values for monocyte migration averaged 47 (+2.7) and for neutrophil migration averaged 62 (±7.7). All assays were done in triplicate. RESULTS Fig. 1 illustrates the relationship between chemotactic activity and increasing platelet factor 4 concentrations. Cell migration toward platelet factor 4 occurred at platelet factor 4 concentrations of 1 pug/ml and increasing concentrations ofplatelet factor 4 to 5 ,ug/ml resulted in increased monocyte and neutrophil migration. At 5 pug/ml, the migration response of test cells to platelet factor 4 nearly equaled (or fully equa...

Some human chronic dermal wounds treated with recombinant platelet-derived growth factor-BB (rPDGF-BB) show increased healing coupled with fibroblast activation and granulation tissue formation. To determine whether endogenous PDGF is associated with healing and nonhealing dermal ulcer phenotypes, we developed monoclonal antibodies capable of recognizing the three isoforms of PDGF, AA, AB, and BB dimers, and capable of discriminating between two alternatively spliced A chain transcripts. We detected little PDGF isoform expression in normal skin and in nonhealing dermal ulcers. In contrast, in surgically created acute wounds and chronic ulcers treated with rPDGF-BB, markedly upregulated levels of PDGF-AA (long form) were found. In both types of wounds, increased PDGF-AA was detected primarily in capillaries and fibroblasts, although in rPDGF-BB-treated chronic wounds, widespread expression of PDGF-AA was somewhat delayed. With continued treatment, the long form of PDGF-AA, which can preferentially bind extracellular matrix, was expressed only in capillaries, while fibroblasts began synthesizing the short form of PDGF-AA. Within capillaries, all endothelial cells and varying numbers of pericytes and smooth muscle cells contained PDGF-AA. In all wounds, macrophages and keratinocytes were not a major contributor. While PDGF-BB and PDGF-AB were present in a minority of healing wounds, they were usually present at lower levels than PDGF-AA. PDGF-18 receptors, which bind only PDGF-BB and not other isoforms, were found in normal skin and granulation tissue, providing a molecular basis for treating human chronic wounds with exogenous rPDGF-BB. (J. Clin.

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