Modulation of fibroblast functions by interleukin 1: increased steady-state accumulation of type I procollagen messenger RNAs and stimulation of other functions but not chemotaxis by human recombinant interleukin 1 alpha and beta.

Interleukin-1 (IL-1) is synthesized by and released from macrophages in response to a variety of stimuli and appears to play an essential role in virtually all inflammatory conditions. In tissues of mesenchymal origin (e.g., cartilage, muscle, bone, and soft connective tissue) IL-1 induces changes characteristic of both destructive as well as reparative phenomena. Previous studies with natural IL-1 of varying degrees of purity have suggested that it is capable of modulating a number of biological activities of fibroblasts. We have compared the effects of purified human recombinant (hr) IL-1 alpha and beta on several fibroblast functions. The parameters studied include cell proliferation, chemotaxis, and production of collagen, collagenase, tissue inhibitor of metalloproteinase (TIMP), and prostaglandin (PG) E2. We observed that hrIL-1s stimulate the synthesis and accumulation of type I procollagen chains. Intracellular degradation of collagen is not altered by the hrIL-1s. Both IL-1s were observed to increase the steady-state levels of pro alpha 1(I) and pro alpha 2(I) mRNAs, indicating that they exert control of type I procollagen gene expression at the pretranslational level. We found that both hrIL-1 alpha and beta stimulate synthesis of TIMP, collagenase, PGE2, and growth of fibroblasts in vitro but are not chemotactic for fibroblasts. Although hrIl-1 alpha and beta both are able to stimulate production of PGE2 by fibroblasts, inhibition of prostaglandin synthesis by indomethacin has no measurable effect on the ability of the IL-1s to stimulate cell growth or production of collagen and collagenase. Each of the IL-1s stimulated proliferation and collagen production by fibroblasts to a similar degree, however hrIL-1 beta was found to be less potent than hrIL-1 alpha in stimulating PGE2 production. These observations support the notion that IL-1 alpha and beta may both modulate the degradation of collagen at sites of tissue injury by virtue of their ability to stimulate collagenase and PGE2 production by fibroblasts. Furthermore, IL-1 alpha and beta might also direct reparative functions of fibroblasts by stimulating their proliferation and synthesis of collagen and TIMP.

Human skin collagenase in recessive dystrophic epidermolysis bullosa. Purification of a mutant enzyme from fibroblast cultures.

Recessive dystrophic epidermolysis bullosa, a genodermatosis characterized by dermolytic blister formation in response to minor trauma, is characterized by an incresaed collagenase synthesis by skin fibroblasts in culture. Since preliminary studies of partially purified recessive dystrophic epidermolysis bullosa collagenase suggested that the protein itself was aberrant, efforts were made to purify this enzyme to homogeneity, so that detailed biochemical and immunologic comparisons could be made with normal human skin fibroblast collagenase. Recessive dystrophic epidermolysis bullosa skin fibroblasts obtained from a patient documented to have increased synthesis of the enzyme were grown in large scale tissue culture and both serum-free and serum-containing medium collected as a source of collagenase. The recessive dystrophic epidermolysis bullosa collagenase was purified to electrophoretic homogeneity using a combination of salt precipitation, ion-exchange, and gel-filtration chromatography. In contrast to the normal enzyme, the recessive dystrophic epidermolysis bullosa collagenase bound to carboxymethyl-cellulose at Ca(2+) concentrations at least 10 times higher than those used with the normal enzyme. Additionally, this enzyme was significantly more labile to chromatographic manipulations, particularly when serum-free medium was used. However, rapid purification from serum-containing medium yielded a preparation enzymatically equivalent to normal human skin collagenase. Like the normal enzyme, the recessive dystrophic epidermolysis bullosa collagenase was secreted as a set of two closely related zymogens of approximately 60,000 and approximately 55,000 daltons that could be activated by trypsin to form enzymically active species of approximately 50,000 and approximately 45,000 daltons, respectively. Amino acid analysis suggested slight variations between the normal and recessive dystrophic epidermolysis bullosa collagenases. Cyanogen bromide digests demonstrated peptides unique to the enzyme from each source. The recessive dystrophic epidermolysis bullosa proenzyme was significantly more thermolabile at 60 degrees C than the normal, a finding that correlated with an approximate fourfold decrease in the affinity of the mutant enzyme for Ca(2+), a known activator and stabilizer of human skin collagenase. Aside from the altered affinity for this metal cofactor, kinetic analysis of the structurally altered recessive dystrophic epidermolysis bullosa collagenase revealed that its reaction rates and substrate specificity for human collagen types I-V were identical to those for the normal enzyme. Likewise, enzymes from both sources displayed identical energies of activation and deuterium isotope effects. Antisera were raised to the normal and putatively mutant procollagenases respectively, and, although they displayed a reaction of identity in double diffusion analysis, immunologic differences were present in enzyme inhibition and quantitative precipitation studies. These studies indicate that recessive dystrophic epidermolysis bullosa is characterized by the increased synthesis of an enzymically normal, but structurally aberrant, collagenase.

EGF and TGF alpha influence in vitro lung development by the induction of matrix-degrading metalloproteinases.

Remodeling of the extracellular matrix by matrix-degrading metalloproteinases (MMPs) has been implicated in the early morphogenesis of branched organs. Growth factors such as EGF and TGF alpha are known to regulate the expression of MMPs in a variety of systems. We therefore examined the effects of EGF, TGF alpha, and collagenase upon in vitro branching of the embryonic lung. Lung rudiments from 11.5 day post coitum mice underwent extensive growth and repetitive branching during a 3-day period in organ culture. Lungs treated with EGF or TGF alpha were larger than controls, yet displayed fewer branches along with markedly dilated end buds which lacked clefts, indicating that these growth factors inhibit normal lung branching. Addition of purified mammalian collagenase to lung cultures similarly inhibited epithelial branching and produced end bud enlargement. In addition, gelatin-substrate enzymography of the conditioned medium from EGF- and TGF alpha-treated lungs revealed a marked induction of a metalloproteinase activity which most likely corresponds to the 72kDa type IV collagenase/gelatinase which degrades basement membrane collagens. Lungs maintained in the presence of both TGF alpha and TIMP, a specific inhibitor of MMPs, branched repeatedly and displayed normal, narrow end buds as seen with controls, suggesting that TIMP is capable of preventing or reversing the observed growth factor mediated effects upon lung branching. Taken together, these results provide evidence that the growth factors EGF and TGF alpha guide lung development, at least in part, by inducing the expression of matrix-degrading metalloproteinases.

Parathyroid hormone-induced resorption in fetal rat limb bones is associated with production of the metalloproteinases collagenase and gelatinase B.

The role of matrix metalloproteinases in parathyroid hormone (PTH)-induced bone resorption was assayed using a fetal rat limb bone culture system. Cotreatment of bones with PTH and recombinant inhibitor of metalloproteinases, TIMP-1, in vitro, inhibited the PTH-stimulated 45Ca release from the limb bones without affecting beta-glucuronidase release. TIMP-1 was fully effective when added during only the final 24 h of a 72 h culture with PTH but was ineffective when added for only the first 24 h of the 72 h culture. In contrast, calcitonin (CT) was effective when added for either the first 24 or the final 24 h of the culture. Using in situ hybridization, the mRNA for collagenase was detected in mononuclear cells of cultured bone. Treatment of the bones with PTH resulted in an increase in the number of cells producing collagenase mRNA, some of which had osteoclastic morphology, PTH also caused a dramatic induction of the mRNA for the 92-kD gelatinase B metalloproteinase in both mononuclear and osteoclastic cells. There was no detectable mRNA for the metalloproteinases stromelysin-1, stromelysin-2, or matrilysin in PTH-treated or control cultures. These results suggest that PTH-induced bone resorption is mediated, at least in part, by the induction of collagenase and gelatinase B mRNA in bone cells.

Immunolocalization of collagenase and TIMP in healing human burn wounds.

Degradative events in remodeling connective tissues are mediated through the actions of one or more members of the matrix metalloproteinase family. Conversely, members of the tissue inhibitors of metalloproteinase (TIMP) family act to attenuate proteolysis. Because collagenase and TIMP are rapidly secreted into the extracellular matrix following their biosynthesis and may not remain near their cell of origin, we undertook an immunohistochemical examination of human burn injuries to establish the distribution of these proteins during acute wound repair. Immunostaining for collagenase and TIMP was markedly increased within the wound bed but not in adjacent regions of histologically normal skin. Immunoreactive collagenase was first noted at the eschar-dermal interface by day 3 after injury and became very prominent in the dermis from day 5 to day 17. By day 5, focal patches of immunoreactive collagenase were found at the epidermal-dermal junctions at the wound margins. Within the wound bed, intense staining for collagenase was noted in the connective tissue surrounding the surviving epithelial appendages and around blood vessels. Immunoreactive TIMP was detected by day 2 both in the dermis and the overlying eschar but rapidly assumed the same interfacial pattern as described for collagenase. Staining for TIMP was only sporadically found at the dermal-epidermal margins and surrounding surviving epithelial appendages. Like collagenase, TIMP was prominently localized about vascular structures. These studies demonstrate that, in acute wounds, immunoreactive collagenase and TIMP are generally increased throughout the area of injury but particularly so at interface zones including eschar-dermis, epidermis-dermis, appendages-dermis, and around vascular structures.

Localization of mRNAs representing interstitial collagenase, 72-kda gelatinase, and TIMP in healing porcine burn wounds.

The process of wound healing sets in motion a complex and dynamic series of events, which includes the remodeling of the extracellular matrix. Degradation of matrix macromolecules is mediated through the actions of the matrix metalloproteinase family. Conversely, the actions of this enzyme family are regulated by tissue inhibitors of metalloproteinases (TIMPs). In this study, we have developed riboprobes derived from human cDNAs representing collagenase, 72-kDa gelatinase, and TIMP and have found them to be sufficiently specific and sensitive for use in in situ hybridization studies of porcine burn wounds. Expression of these mRNAs, although not seen in uninjured skin, was found to be a predictable and locally distinct event in wound repair. Transcripts for collagenase and TIMP but not 72-kDa gelatinase were detected at the resurfacing epithelial margin; label was also detected in and around follicular epithelium within the wound bed. Transcripts for both metalloenzymes and TIMP were found throughout the viable dermis and subcutaneous tissues underlying the wound bed. However, expression of 72-kDa gelatinase was most prominent in the superficial dermis adjacent to the resurfacing epidermis at the wound margin. Collagenase and TIMP transcripts were particularly prominent in a perivascular pattern in the dermis and in the connective tissue network surrounding adipocytes in the subcutaneous zone. Numerous cell types appeared to be involved, including keratinocytes, fibroblasts, macrophages, and endothelial cells. Future exploitation of this porcine thermal injury model is likely to provide information about the spatial and temporal patterns of matrix metalloproteinase and TIMP expression in cutaneous wound healing.

Elevated levels of human collagenase inhibitor in blister fluids of diverse etiology.

Blister fluids from a variety of bullous disorders were examined for the presence of human collagenase inhibitor. A protein immunologically identical to the collagenase inhibitor produced by human skin fibroblasts was found in high concentrations within bullae of diverse etiologies. Levels of collagenase inhibitor in blister fluids ranged from 0.9-12.5 micrograms/ml, averaging 4.9 micrograms/ml. The mean values were 3- to 4-fold greater than those present in the sera of corresponding patients and exceeded plasma levels by 6- to 8-fold. The time course of collagenase inhibitor accumulation in blister fluid was studied using heat- and suction-induced bullae. The concentration in newly formed blisters was approximately 0.5 micrograms/ml, virtually identical to plasma inhibitor levels, and remained constant for approximately 4 h. Inhibitor concentrations then rose rapidly, reaching peak values of approximately 6 micrograms/ml after 48 h. We speculate that the role of this inhibitor in blister fluid involves the inhibitions of active proteinases within the bulla cavity and may occur to limit the extent of blister formation or to assist in wound repair.

Constitutive production of procollagenase and tissue inhibitor of metalloproteinases by human keratinocytes in culture.

Production of procollagenase and tissue inhibitor of metalloproteinases was demonstrated in human keratinocyte cultures. The two proteins were immunoprecipitated from keratinocyte-conditioned medium with antibodies to human dermal fibroblast collagenase and tissue inhibitor of metalloproteinases and quantitated with enzyme-linked immunosorbent assays. Treatment of the keratinocytes with the phorbol ester, 12-0-tetradecanoylphorbol-13-acetate, produced a six to 34-fold increase in procollagenase synthesis and secretion but only a threefold increase in the production of tissue inhibitor of metalloproteinases. Collagenase and tissue inhibitor of metalloproteinases mRNAs were present in normal keratinocytes, were the same size as their fibroblast counterparts, and both increased in response to treatment with 12-0-tetradecanoylphorbol-13-acetate. These data suggest that remodeling of type I collagen may be an important function of human keratinocytes in vivo.

Enhanced synthesis of collagenase by human keratinocytes cultured on type I or type IV collagen.

Human keratinocytes in culture are known to produce collagenase. As part of studies to ascertain the physiologic stimuli for collagenase production by keratinocytes, we wanted to determine whether extracellular matrix could modulate the production of collagenase in vitro. Immunoprecipitable collagenase from the conditioned medium of cells grown on different types of matrix was measured. Metabolically labeled human keratinocytes were cultured in 0.1 mM calcium in serum-free medium on colloidal gold-coated coverslips plus type IV collagen, type I collagen, or laminin or in the absence of matrix. Immunoprecipitation of the conditioned medium with anti-collagenase antiserum was performed and the immunoprecipitates were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, fluorography, and densitometry. The keratinocytes cultured on type IV or type I collagen produced more collagenase than did those cultured on laminin or in the absence of matrix. This effect did not reflect a general increase in secreted proteins, because the production of tissue inhibitor of metalloproteinase, or TIMP, did not increase under the same conditions. Phagocytosis of the gold salts by the keratinocytes migrating on types I or IV collagen did not account for the increased collagenase produced by these cells since the effect persisted in the absence of the colloidal gold and phagocytosis of latex beads did not augment collagenase production.

Singlet oxygen may mediate the ultraviolet A-induced synthesis of interstitial collagenase.

Singlet oxygen has been postulated to be generated by Ultraviolet (UV) A irradiation of mammalian cells. We studied the role of singlet oxygen in the downstream signaling of the complex UV response leading to the induction of matrix-metalloproteinase-1 (interstitial collagenase/MMP-1). Exposure of cultured human fibroblasts to singlet oxygen, generated in a dark reaction by thermodissociation of the endoperoxide of the disodium salt of 3,3'-(1,4-naphthylidene) dipropionate (NDPO2) induced collagenase mRNA steady state levels in a dose dependent manner. The increase in collagenase expression after singlet-oxygen exposure generated with 3 mM NDPO2 was equivalent to that observed with UVA at a dose rate of 200-300 kJ/m2 and developed in a similar time course. In contrast, mRNA levels of TIMP-1, the specific tissue inhibitor of metalloproteinases, remained unchanged. Indirect evidence for the role of singlet oxygen in the UVA induction of collagenase comes from studies using singlet oxygen enhancer or quencher. Accordingly, incubation in deuterium oxide, an enhancer of singlet-oxygen lifetime, led to an additional increase in steady-state levels of collagenase mRNA after exposure to NDPO2 or to UVA irradiation. In contrast, sodium azide, a potent quencher of singlet oxygen, almost totally abrogated the induction of collagenase after exposure of fibroblasts to NDPO2 or to UVA irradiation. Similar results were obtained in studies of the proteins by radioimmunoprecipitation of MMP-1 and TIMP-1 using specific antibodies. Collectively, our data provide circumstantial evidence that singlet oxygen mediates the UVA induction of collagenase in vitro, whereas it does not exert any effect on TIMP-1 synthesis. The unbalanced synthesis of interstitial collagenase may contribute to the connective tissue damage in vivo related to photoaging and other photocutaneous disorders.

Increased immunostaining of collagenase and TIMP in eruptive xanthoma.

Remodeling of the extracellular matrix is an important function of interstitial collagenase. The activity of this enzyme forms the initial and rate limiting step in collagen degradation; moreover, this enzyme appears representative of a family of connective tissue metalloproteinases. Conversely, a widely distributed glycoprotein, tissue inhibitor of metalloproteinases (TIMP), may be an important regulator of matrix degradation. To study the roles of collagenase and TIMP in pathologically altered dermal connective tissue, immunohistochemistry was used to localize collagenase and TIMP in an eruptive xanthoma, a chronic tuberous xanthoma, and normal skin. Normal skin and the chronic tuberous xanthoma showed mild diffuse staining of both proteins throughout the dermis. In contrast, intense dermal staining of both collagenase and TIMP was present in the eruptive xanthoma. Thus, the marked accumulation of lipid in dermal macrophages was associated with a significant increase in matrix collagenase and TIMP. This increase may reflect direct production of these two proteins by macrophages. Alternatively, it may be due to increased production by fibroblasts stimulated by macrophage-derived cytokines. The balance of degradative and inhibitory activities in the extracellular matrix may regulate the extent and nature of dermal remodeling.

A comparison of scar revision with the free electron and carbon dioxide resurfacing lasers.

Laser scar revision was studied to measure the effects of targeting extracellular matrix protein versus tissue water on scar revision. We compared the free electron laser used at 7.7 microm (the amide III protein absorption band) to the carbon dioxide (CO2) laser and dermabrasion.Nude mice (n = 40) that had rejected skin grafts on their dorsal surface and developed mature scars were used as a model for scar revision. One-half of each scar was revised with either the free electron laser at 7.7 microm (32 to 38 mJ, nonoverlapping pulses delivered with a computerized adjustable pattern generator at 30 Hz, and two to three passes), a 100-microsec CO2 resurfacing laser (500 mJ, 5.0 Hz, and two to five passes), or dermabrasion. The untreated portion of each scar served as an internal control. Evaluation was by measurement of the clinical size of the scar using photography with quantitative computer image analysis to compare the data and histology to evaluate the quality and depth of the scars. The free electron laser at 7.7 microm was significantly better than the CO2 laser and dermabrasion for scar size reduction (p < 0.046 and p < 0.018). The CO2 laser and a highly skilled dermabrader were not statistically significantly different (p < 0.44). The result seen with less skilled dermabraders was significantly worse than all other methods (p < 0.009). The free electron laser at 7.7 microm, which is preferentially absorbed by the proteins of the extracellular matrix, provided better scar reduction than the CO2 resurfacing laser and dermabrasion. Dermabrasion by a skilled operator resulted in improvement similar to the results obtained with the CO2 resurfacing laser, but less skilled operators had significantly poorer results.

Expression of matrix metalloproteinases and tissue inhibitor of metalloproteinases in head and neck squamous cell carcinoma.

Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes implicated in the invasion and metastasis of many cancers. In situ hybridization techniques were used to reveal sites of expression of collagenase (MMP-1), gelatinase 72 kd (MMP-2), gelatinase 92 kd (MMP-9), and tissue inhibitor of metalloproteinase-1 (TIMP-1) in head and neck carcinomas (N = 21). Both TIMP-1 and gelatinase 72 kd were expressed in nearly all tumors, whereas the expression of collagenase and gelatinase 92 kd showed variability. Tumor-associated expression of MMPs was strongest in stromal cells near advancing margins. No differences in expression levels were detected between primary and metastatic sites. This paper reviews the literature and discusses the significance and possible implications of MMPs in head and neck squamous cell carcinoma.

Human fibroblast tissue inhibitor of metalloproteinases: glycosylation and function.

The glycosylation of human fibroblast tissue inhibitor of metalloproteinases and procollagenase was examined in vivo using tunicamycin B2 and in vitro using Peptide: N-glycosidase F. In the presence of tunicamycin B2, unglycosylated inhibitor continues to be synthesized and secreted at normal or increased rates. The protein core of this collagenase inhibitor has an apparent Mr of 21,000 and possesses at least two oligosaccharide linkage sites as evidenced by the accumulation of a single 25,000 dalton intermediate. Collagenase inhibitor deglycosylated by Peptide: N-glycosidase also has an apparent molecular weight of 21,000 daltons; furthermore, deglycosylated inhibitor continues to block the activity of collagenase at a 1:1 molar stoichiometry and does not differ from the native glycoprotein in its resistance to tryptic degradation. Using these same two reagents, the characteristic doublet of human fibroblast procollagenase was found to result from glycosylation in the upper (60,000 dalton) form. Secretion of procollagenase was significantly inhibited in the presence of tunicamycin B2.

Human skin fibroblast collagenase inhibitor. Purification and biochemical characterization.

Human skin fibroblasts maintained in cell culture secrete a collagenase inhibitor which has been purified to homogeneity from serum-containing culture medium using a combination of salt precipitation, and ion-exchange, phenylboronate-agarose, gel filtration, and high pressure liquid chromatography. The pure inhibitor retained full activity and exhibited a 1:1 molar inhibition of collagenase. A single band of Mr = 28,500 was seen on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition was remarkable for the presence of 24 half-cystine residues. No free sulfhydryls were present and the resultant 12 disulfide bridges may account for the remarkable stability of this protein to extremes of pH, temperature, pressure, as well as to denaturing agents. A total of 13 hexose residues/molecule were found. NH2-terminal sequence analysis revealed the presence of a single polypeptide chain and the first 23 residues were identified. A monospecific antibody was produced which abolished the functional activity of the inhibitor. Fibroblast inhibitor was found to migrate with the beta-globulins by immunoelectrophoresis. A chromatographically and immunologically identical collagenase inhibitor was partially purified from human serum, suggesting the possibility that the fibroblast-derived inhibitor and the previously reported serum beta 1-anticollagenase (Woolley, D. E., Roberts, D. R., and Evanson, J. M., (1975) Biochem. Biophys. Res. Commun. 66, 747-754) are similar, if not identical, proteins. The fibroblast collagenase inhibitor was found to be clearly distinct from other collagenase inhibitors of leukocyte and serum origin.

Human skin fibroblast collagenase inhibitor. Comparative studies in human connective tissues, serum, and amniotic fluid.

In order to gain insight into the biological significance of a collagenase inhibitor secreted by human skin fibroblasts, we examined various human connective tissues and body fluids for such a protein. The inhibitors found in these tissues were compared immunologically to skin fibroblast inhibitor by Ouchterlony analysis and by the development of a highly specific enzyme-linked immunosorbent assay (ELISA). Using this ELISA, cell cultures of human skin fibroblasts, corneal fibroblasts, gingival fibroblasts, and adult and fetal lung fibroblasts secreted similar amounts of immunoreactive inhibitor protein. Each culture medium displayed a reaction of immunologic identity with skin fibroblast inhibitor when examined in Ouchterlony gel diffusion. In testing for functional inhibitory activity, the same 1:1 stoichiometry of collagenase inhibition was observed in each culture medium that characterizes the human skin inhibitor. Other mesodermally derived human cell types, including human fetal osteoblasts, uterine smooth muscle cells, fibrosarcoma cells, and explants of tendon and articular cartilage behaved in the same manner as the fibroblast cultures. Because collagenase inhibitors with biochemical similarities to skin fibroblast inhibitor have been described in serum and in amniotic fluid, we also examined these sources of inhibitory proteins. The data indicate that both serum and amniotic fluid contain collagenase inhibitors which are immunologically and functionally identical with the skin fibroblast inhibitor. The concentration of inhibitor in serum, as measured by ELISA assay, is 1.03 +/- 0.27 micrograms/ml. The results suggest that collagenase inhibitors which are functionally equivalent and immunologically identical with human skin fibroblast collagenase inhibitor are synthesized by many, if not all, fetal and adult mesodermal tissues in the human organism. The inhibitor apparently gains access to certain body fluids such as serum and amniotic fluid. This inhibitor protein may, therefore, function in the regulation of collagen degradation in most human connective tissues.

The influence of cell culture variables on human collagenase inhibitor expression.

This study investigated the modulation of human collagenase inhibitor expression by a variety of cell culture variables. Inhibitor production was found to be largely invariant with respect to the concentration of serum incorporated into culture medium. Similarly, environmental pH failed to affect inhibitor expression over the pH range of 6.8-8.2. When inhibitor production was examined as a function of cell culture density, synthesis of this protein per cell was greatest during logarithmic growth and early confluence, but even in late post-confluent cultures levels approaching 50% of maximal were routinely observed. When basal levels of collagenase and inhibitor were compared in 8 different normal human skin fibroblast cell lines, inhibitor production varied by only 2-fold, whereas collagenase levels displayed a range exceeding 20-fold. Thus, despite manipulations in the presence or absence of serum and even across different cell lines, inhibitor production seemed to be marked most conspicuously by its constancy. The kinetics of inhibitor secretion into culture medium were also examined. Whether cultured in the presence or absence of serum, inhibitor levels reached maximal values in the medium after 24-48 hours of incubation and remained constant thereafter. Interestingly, assessment of intracellular versus extracellular quantities of inhibitor demonstrated that there was no significant intracellular storage of this protein. Thus, the data suggest that human collagenase inhibitor is a secretory protein which is rapidly exported into the extracellular space without significant accumulation intracellularly.