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Experimental Cutaneous Calcinosis: a Histopathologic and Histochemical Study*
EXPERIMENTAL CUTANEOUS CALCINOSIS: A HISTOPATIHOLOGIC AND HISTOCHEMICAL STUDY* WAINE C. JOHNSON, M.D., P. DONALD FORBES, Pn.D., JAMES H. GRAHAM, M.D. AND HOWARD R. GRAY, M.D.
Tissue calcification is a dynamic process and the end stage, when studied by morphological, histochemical, or chemical methods, yields only
was fixed in neutral buffered formalin for paraffin-
limited information concerning the pathogenesis.
hours, and at 3, 4, 5, and 6 days after injection. The number of animals and the time period of obtaining tissue after treatment with DHT and egg white were as follows: 18 hours, 2; 24 hours, 3; 32 hours, 2; 40 hours, 1; 48 hours, 2; 56 hours, 1; 3 days, 1; 4 days, 1; and 5 days, 1. Specimens
embedding. A total of 34 animals treated with DHT and iron dextran were studied and tissue
Because of the difficulty in obtaining adequate tissue specimens representing documented early phases of pathologic calcinosis in humans, it has been necessary to study experimental cal-
specimens were obtained at 18, 24, 32, 40 and 48
cinosis in laboratory animals. Selye (1) has from animals treated with DHT and plucking were taken at 24 hours, 32 hours, 48 hours, 56 hours,
described excellent methods of inducing experimental cutaneous calcinosis in the rat. Although Selye and others have published detailed biolog-
3 days and 4 days (1 animal used F or each interval).
Controls consisted of normal animals and animals treated with only DHT, iron dextran or egg white.
ical data concerning experimental calcinosis Normal skin from the back of 4 animals was ("calciphylaxis"), adequate histoehemical and studied. Tissue specimens were obtained from 7 animals treated with DHT alone, and these were
histopathological features of early developmental stages have not been described. In this paper we shall report our observations in a preliminary histopathologic and histochemical study of experimental cutaneous calcinosis in the rat.
taken at intervals of 1, 2, 3, and 5 days after
ingestion of DHT. Specimens from animals (1 each interval) treated with iron dextran were taken at 24, 32, 48, and 56 hours, and at 3, 4, and 5
days. Tissue specimens from control animals
treated with egg white were obtained at the same intervals as the iron dextran controls. Formalin-fixed, paraffin-embedded tissue secMATERIAL5 AND METHODS tions were prepared by the following methods: The animals used were female Lewis albino hematoxylin and eosin; the colloidal iron technic, rats, each weighing approximately 100 grams, ex- with and without bovine testicular hyaluronidnse cept for 1 group of female Wistar rats weighing digestion for 1 hour at 37 C; the alcian blue stain approximately 200 grams. Oral dihydrotachysterol at pH 2.5 and 0.4; the aldehyde-fuchsin stain at (DHT), 1 mg in 0.5 ml corn oil, was used as the pH 1.7 and 0.4; periodic acid-Schiff (PAS) reacsensitizer, and an elicitor was given 24 hours later. tion, with and without diastase digestion; potasElicitors used were iron dextran, egg white and sium ferrocyanide and hydrochloric acid method plucking. Two ml of a 17% solution of iron dcx- for iron; and the alizarin red S and the von Kossa tran in distilled water, or 5 ml of a 50% solution methods for calcium. The technics followed and of egg white in distilled water, was inj ected the basis for our interpretation of a positive reacsubcutaneously in the midline of the back. The tion, except for the von Kossa stain, have been animals were lightly anesthetized with ether at the reported (2, 3). The von Kossa stain (4) is based time of injection or plucking (mechanical dep- on the combination of silver with anion salt which ilation). Tissue specimens taken from the midline may be carbonate, phosphate, oxalate, sulfate, of tbe back were immediately frozen with dry chloride, or sulfocyanide, and reduction to meice and used for cryostat-sectioning. Adjacent skin tallic silver by exposure to light. Those salts remaining in tissue sections after routine processing
* From
the Skin and Cancer Hospital of Phila- are almost always due to calcium. Urates and uric acids are the only serious source of error and these
delphia, Department of Dermatology, Temple
compounds can be removed by their solubility
University School of Medicine, Philadelphia, Pa.
in lithium carbonate solution. Procedures to demThis investigation was supported in part by re- onstrate calcium, iron, and acid mucopolysacsearch training grant no. 2A-5289 (C2), from the charides were then performed. Non-specific alkaline 19107.
National Institute of Arthritis and Metabolic
Diseases, and grant no. CA 07478, from the Na- phosphatase preparations using acetone-fixed, cryotional Cancer Institute, U. S. Public Health Serv- stat-sections were performed using naphthol ASMX phosphate (Sigma) and fast blue RH or red ice, Bethesda, Maryland 20014. Presented at the Twenty-fifth Annual Meeting violet LB salts as substrate and coupling dyes of The Society for Investigative Dermatology, respectively. Sections mounted on cover slips, or free sections, were incubated 10 to 15 minutes in Inc., San Francisco, Calif., June 22, 1964. 453
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THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
the substrate at pH 8.6 and some were counterstained with toluidine blue, aldehyde-fuehsiu, or
These reactions arc consistent with a sulfated acid mucopolysaccharide such as heparin. Alalizarin red S. Sections were mounted in glycerolgelatin or were dehydrated and passed quickly though purified heparin has been reported to through xylene to Permount. Occasional cryostat- be PAS negative, (5) protein complexes or sections were stained with alizarin red S to rule other material present in the granules may out the possibility of the calcium being leeched give rise to the positive PAS reaction. Extraout by formalin-fixation or paraffin-embedding. cellular material located in the dermal anagen Acid mucopolysaccharides and mucopolysaccharide-protein complexes were identified by the hair papillac was colloidal iron positive, hyalucolloidal iron, alcian blue, aldehyde-fuchsin, and ronidase resistant, alcian blue positive at pH PAS techuics. Mast cells were most readily identi- 2.5 and 0.4, aldehydc-fucbsin positive at pH fied by the aldehyde-fuchsin stain but were also 1.7 and 0.4, and PAS negative. These reactions demonstrated by alcian blue, colloidal iron, and indicate a sulfated acid mucopolysaccharidc and PAS methods. Elastic fibers were demonstrated by the aldehyde-fucbsin stain. Iron was identified by would be consistent with those of chondroitin the ferrocyanide iron stain and calcium was dem- sulfate B. The outer root sheath of the anagen onstrated by alizarin red S and von Kossa methods. hair contained a small amount of reacting Combination methods used to demonstrate multi- material which was colloidal iron positive, alple elements in the same tissue section included methods for: iron, mast cells, and calcium; calcium cian blue positive at pH 2.5 and negative at and alkaline phosphatase and alkaline phosphatase 0.4, aldehydc-fuchsin negative, and PAS negative
and mast cells (demonstrated by toluidine blue or aldehyde-fuchsin). RESULTS AND INTERPRETATION
Normal Skin: Only those histological and histochemical features pertinent to this study will be described. Prominent differences of rat skin from that of human include the relative thinness of the epidermis, absence of sweat glands, a well developed panniculus carnosus muscle (PCM), a layer of fat cells above the PCM and the alterations associated with the hair cycle. The upper third of the dermis is composed chiefly of collagen fibers aud is asso-
after diastase digestion. Most of this material was hyaluronidasc-resistant. Due to the small amount of material, histochemical characterization was not entirely satisfactory. Graham et al.
(6) have demonstrated the presence of hyaluronic acid in the outer root sheath of human hair follicles. The cortex of the hair, especially in the middle and lower portions, reacted to the aldehydc-fuchsin stain at pH 1.7 and to a lesser
extent at pH 0.4. Since other stains for acid mucopolysaccharides were negative or only weakly positive in the hair cortex, the nature of the aldehyde-fuchsin reactive material is unknown. Abul-Haj and Rinehart (7) suggested
ciated with a relatively greater number of that aldehyde-fuchsin has an affinity for organic
fibrocytes than that seen in human skin. The middle third, in the anagen phase, is composed
sulfur containing compounds in addition to sulfated acid mucopolysaccharidcs. The high sul-
of fat cells with some collagen fibers inter- fur content of hair may be the basis for the
spersed between the fat cells, and contains the affinity observed. Only a small amount of acid middle aud lower portions of the hair follicle. mucopolysaccharidc could be demonstrated in This zone is greatly diminished in the telogen the extracellular interfibrillar ground substance.
phase, and the resting follicle is contained
Most of tlus material was located between
primarily within the collagen of the upper derthe fat cells and about the hair follicles in the mis. The lower third of the dermis is composed of the PCM and a narrow zone of collagen zunc above the PCM. This extracellular interfibers beneath the PCM, separating it from the fibrillar material showed reactions indicating hysubcutaneous tissue. Mast cells were located ad- aluronic acid, i.e. colloidal iron positive, hyjacerit to blood vessels at all levels in the dermis aluronidase labile, alcian blue positive at pH
and the number, even at concentrated sites, 2.5 and negative at pH 0.4, aldehydc-fucbsin rarely exceeded ten per high power field. Mast negative, and PAS negative. The aldehyde-
fuchsin stain demonstrated elastic fibers in the stain at pH 1.7 and 0.4, the alcian blue stain upper corium, an absence of elastic fibers in at pH 2.5 and 0.4, the colloidal iron technic, the mid-dermis, a few fibers immediately above and the PAS method. Hyaluronidase digestion the PCM and a narrow dense band of fibers did not remove the colloidal iron reactivity. immediately beneath the PCM. Stains for iron cell granules reacted with the aldchyde-fuchsin
455
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FIG. 1. Normal skin. Alkaline phosphatase reaction. A. Capillaries, sebaeeous gland cells, dermal
hair papillae, and a zone about the lower portion of the follicles give a positive reaction. X 50. B. Intense
alkaline phosphatase activity is present in the dermal papilla of an anngen follicle and along a narrow zone extending about the outer root sheath in the lower part of the follicle. X 66.
and calcium were negative. Alkaline phospha- traeellular and intracellular in the zone above tase activity was observed in the capillary- the PCM, an occasional area between the muscle endothelium and sebaeeous gland cells (Fig. 1A), bundles, and a large amount both extraeellular and intense activity in the dermal hair papillae and intracellular beneath the PCM. Technies with a narrow zone extending about the outer showing both iron and mast cells in the same root sheath in the lower portion of the follicle tissue section revealed the mast cells did not (Fig. lB). No alkaline phosphataso activity was contain iron. The PAS technic showed a brownish-red reaction at sites of intracellular demonstrated in mast cells or fibroeytes. DHT Treated: Except for the number of and extraeellular iron. The reactivity presumafibroeytes and mast cells, no histiologie or histo- bly is due to dextran, although the possibility of chemical changes from that seen in normal skin recombination of iron with other tissue elements was observed in specimens taken 1 to 5 days must be considered. A few to a moderate numafter administration of DUT. An increased num- ber of polymorphonuclear leukocytes, and small ber of mast cells and fibrocytes was observed in round cells, as well as increased numbers of the latter part of the peroid, however, due to fibrocytic and histiocytic cells were present the limited number of specimens studied, this in the dermis and subcutaneous tissue beneath apparent moderate increase in number could the PCM. Specimens taken at 32 hours showed a relatively larger proportion of the iron to be be a chance occurrence.
Iron Dextran Treated: Specimens taken at located intraeellularly. An additional finding was 24 hours showed an increased number of fibro- the presence of an increased amount of extraeytcs and mast cells located principally in the cellular interfibrillar material showing histomiddle of the dermis and beneath the PCM. chemical properties of hyaluronie acid, located Iron was demonstrated to be present within a principally in the middle of the dermis between few fibrocytes in the upper corium, both cx- fat cells and about hair follicles, and beneath
456
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
the PCM. Specimens taken at later intervals
reactions indicate that the material is a non-
showed an increased amount of hyaluronic acid sulfated acid mucopoiysaccharide or acid mucoin the interfibrillar ground substance, and a per- polysaceharide-protein complex and does not sistence of the increased numbers of fibrocytic contain a significant quantity of hyaluronic
acid. Specimens taken at 32 and 48 hours
cells.
Egg White Treated: Specimens taken at 24 showed increased numbers of fibrocytes and hours showed a moderate increase in the num- histiocytes, an increased amount of hyaluronic ber of fibrocytes and mast cells in the middle acid and decreased numbers of inflammatory of the dermis and beneath the PCM. An in- cells beneath the PCM. Only an occasional increased amount of extracellular interfibrillar material showing properties of hyaluronic acid was also present in these areas. A moderate number
flammatory cell was observed above the PCM at
any stage. Specimens taken at 3 and 4 days
continued to show an increased number of mast of eosinophilic polymorphonuclear leukocytes cells and increased amounts of hyaluronic acid and small round cells, in addition to increased located principally in the mid dermis and below numbers of histocytie and fibrocytic cells, were the PCM. The number of fibrocytes was somepresent in the subcutaneous tissue and in the what less in tissue taken on the fourth day. dermis beneath the PCM. Some specimens DHT and Iron Dextran Treated: Specimens taken at 18 hours after injection showed some showed a foreign material (egg white) separating the tissue elements in the subcutaneous tissue. increase in the number of fibrocytes and an This material was colloidal iron psoitive, hy- occasional polymorphonuclear leukocyte in the aluronidase-resistant, alcian blue positive at dermis above the PCM. A moderate number of pH 2.5 and negative at 0.4, aldehyde-fuchsin polymorphonuclear leukocytes, mostly eosinopositive at pH 1.7, PAS positive and diastase phils, and an increased number of fibrocytic, resistant (Fig. 2). Egg white smeared on glass histiocytic, and mononuclear cells were present slides showed identical staining reactions. These in the subcutaneous tissue and in the dermis
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457
EXPERIMENTAL CUTANEOUS CALCINOSIS
beneath the PCM. A relatively large amount of nantly in the zone above the PCM, was demintracellular and extracellular iron was present onstrated. Alkaline phosphatase activity was in the zone above the PCM, occasional iron- observed in fibrocytic cells above as well as becontaining fibrocytic cells were located between low the PCM (Fig. 3). The von Kossa and the muscle bundles, and a moderate amount of alizarin red S technics revealed early caliron was present in the dermis and subcutaneous cification, in 1 specimen, above the PCM in tissue beneath the PCM. Tcchnics for acid muco- the same zone which showed an increased polysaccharides showed an increased amount of amount of hyaluronic acid. Most tissue specihyaluronic acid beneath the PCM in the der- mens taken at 48 hours showed patchy areas of mis and subcutaneous tissue. Alkaline phos- calcification in the mid dermis and in the dermis phatase activity was observed in fibrocytic cells beneath the PCM (Fig. 4). Some specimens located in the dermis beneath the PCM. The showed calcification extending into the upper predominant activity was in the cytoplasm, corium almost to the epidermis. Small patchy although the possibility of activity in the nucleus areas of calcification were occasionally seen could not be completely excluded. The cells between the bundles of the IPCM. A moderate initially appeared at sites near capillaries. Tis- number of fibrocytes containing intracellular sue sections prepared to demonstrate both al- iron, as well as extraccllular iron was present.
Much of the extraccllular iron appeared to be coated on or located within the collagen fibers. Mast cells were decreased in number or absent in calcified areas, but were increased in number between and about the calcified sites. Calcifica-
tion of mast cell granules was not observed in sections prepared for demonstration of both calcium and mast cell granules. An increased
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kaline phosphatase activity and mast cells revealed that the mast cells did not show alkaline phosphatase activity. Specimens taken at 24 hours showed a greater number of fibrocytes and mast cells above the PCM and a smaller number of polymorphonuclear leukocytcs below the PCM than at 18 hours. An increased amount of hyaluronic acid located predomi-
FIG. 3. DHT and iron dcxtraa treated. Alkaline phosphatase reaction. A moderate number of alkaline phosphatasc positive fibrocytic cells arc located above the PCM and a few arc beneath the PCM at 24 hours after injection. )< 55.
458
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
amount of hyaluronic acid in the mid-dermis and
the subcutaneous tissue to the epidermis. Speci-
beneath the PCM was demonstrated by tech- mens taken at 5 and 6 days showed diffuse flies for acid mucopolysaecharides. Some speci- patchy calcified areas throughout the dermis mens showed calcification of areas in the mid- except for some uninvolved areas in the PCM. dermis without calcification in the lower der- Cells were few to absent in the calcified areas. mis beneath the PCM. A large number of Iron remained limited to the middle and lower fibrocytic cells showing alkaline phosphatase dermis and was not present in calcified areas activity were demonstrated in most specimens. in the upper dermis. Some iron appeared coated Alkaline phosphatase positive cells usually were on or absorbed by the collagen fibers whereas not immediately associated with the calcified some remained between the collagen fibers or areas. Specimens taken at 3 and 4 days showed within cells. Alkaline phosphatase positive fibrosimilar features except the calcified areas occu- eytic cells were numerous in the peripheral pied almost the entire dermis in a diffuse but zones of calcification and in adjacent unealeipatchy involvement including a few calcified fled tissue. Reactions for acid mueopolysaeehaareas within the PCM. Alkaline phosphatase rides revealed an increased amount of hyaluronie positive fibrocytie cells were numerous in the acid in most calcified areas. Calcified areas
upper eorium and activity was not entirely did not show a greater PAS reactivity than intracellular (Fig. 5). The extracellular fine the adjacent collagen. A few sections showed granules may have represented a diffusion artifact. The morphology of the collagen fibers in the calcified areas was greatly altered and many small basophilie granules were present in the hematoxylin- and eosin-stained sections. Iron usually remained localized to the mid-dermis even in those areas where the calcium extended from
small areas of necrosis in the superficial dermis with an associated collection of polymorphonuclear leukoeytes.
DHT and Egg White Treated: Specimens taken at 18 hours showed a moderate increase in the number of fibroeytes and histioeytes in the zone above the PCM. Numerous infiam-
Fm. 4. DIIT and iron dextran treated. Alizarin red S stain for calcium. Most tissue specimens taken at 48 hours showed ealcinm deposition in the mid-dermis and in the dermis beneath the PCM. X 50.
459
EXPERIMENTAL CUTANEOUS CALCINOSIS
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FIG. 5. DHT and iron dextran treated. Alkaline phosphatase reaction. At 4 days numerous alkaline phosphatase positive fibrocytic cells were present in the upper dermis. Some activity appeared to be extraeellular, however this might be a diffusion artifact. X 81.
matory cells including eosinophilie polymorpho- present in the upper dermis. Mast cells and
nuclear leukoeytes, small round cells, and an increased number of histiocytes and fibroeytes were present in the dermis below the PCM and in the subcutaneous tissue. Fereign material (egg white) separated normal elements of the
hyaluronie acid were present in increased amounts in the mid dermis and beneath the
PCM. One specimen taken at 48 hours showed calcification limited to the zone above the PCM (Fig. 6). The calcified area showed a marked subcutaneous tissue and showed identical bisto- increase of hyaluronie acid, whereas there was chemical characteristics as that of the material a relatively small amount of hyaluronie acid seen in animals injected with egg white without present below the 2CM (Fig. 7, A and B). DHT treatment. An increased amount of hyalu- Alkaline phosphatase positive fibrocytie cells ronie acid was demonstrated in the zone above were numerous below the PCM and only a the PCM, and a lesser amount between bun- few were present in the calcified area (Fig. 8). dles of the PCM and in the subjacent der- Another specimen taken at 48 hours showed mis. A few to moderate number of alkahne large areas of calcification in the subcutaneous pliosphatase positive fibroeytie cells were present tissue and only small areas of early calcification in the dermis beneath the 2CM. Specimens above the PCM between the fat cells. Teehnies taken at 24 hours showed similar changes for acid mueopolysaeeharides were positive in except that alkaline phosphatase positive cells all areas of calcification. Specimens taken at 3 were present in the mid-dermis as well as be- and 4 days demonstrated extension of calcified neath the 2CM. The number of acute inflam- areas throughout the dermis with the 2CM matory cells in most specimens was less than being least affected. In densely calcified areas, that at 18 hours. Specimens taken at 32 hours colloidal iron reactive material was only partially showed similar features except the number of hyaluronidase-labile. Specimens taken at all alkaline phosphatase positive cells had increased intervals were negative for iron. The connecin the middle and lower dermis and a few tive tissue sheath about hair follicles were cal-
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EXPERIMENTAL CUTANEOUS CALCINOSIS
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F'o. 8. DHT and egg white treated. Alkaline phosphatase reaction. A section adjacent to those seen in Figs. 6 and 7 show several alkaline phosphatase positive fibrocytic cells beneath the PCM but only a few are present in the immediate vicinity of the calcified area. X 55.
cified in the later stages but the dermal papillae positive material in the outer root sheath, an and follicular epithelium usually were not calci- increased number of mast cells and fibrocytie fied.
cells, and a few to a moderate number of
DHT Treated and Plucked: Specimens taken alkaline phosphatase positive fibrocytic cells in at 24 hours showed early anagen follicles. The the upper dermis. No sites of calcification were follicles were distorted, especially in the upper observed. The follicles in both specimens were
half, dilated, and contained loose keratinous in the telogen phase. A specimen taken at material. Fibrocytic cells and mast cells were 56 hours showed distorted anagen follicles. The
increased in number in the upper eorium. collagen bundles and hair follicles in the upper Calcium was demonstrated in some follicles half of the dermis showed minute basophilic near the level of the sehaeeous gland. Loose granules in hematoxylin- and eosin-stained seckeratinous material within follicles and the con- tions. A dense zone of calcification involved the nective tissue sheaths about the follicles were connective tissue sheath about the follicles, most
the predominant anatomical structures calci- dermal hair papillae, and the cortex of the fied. Alkaline phosphatase positive fibrocytie hair within some follicles (Fig. 10, A and B). cells were present in the upper corium and A patchy diffuse calcification was observed in some showed a close relationship to capillaries the upper half of the corium in most areas. adjacent to involved follicles (Fig. 9). Increased Numerous alkaline phosphatase positive fibroamounts of hyaluronie acid were present in the cytie cells were present in the upper eorium mid-dermis. Most of the colloidal iron posi- and in some areas were found in close proxtive material associated with the follicular epi- imity to the hair follicles (Fig. 11). An increased thelium was hyaluronidase-resistant. Specimens amount of interfibrillar ground substance matetaken at 32 and 48 hours showed some distortion rial in the mid-dermis and a lesser amount bein the upper part of the follicles, an increased tween the follicles in the upper dermis showed amount of hyaluronie acid in the mid-dermis, histochemical properties of hyaluronie acid. moderately increased amounts of colloidal iron Most of the colloidal iron positive material
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Fin. 10. DHT and plucked. Von Kossa stain for calcium. A. At 56 hours a dense zone of calcification was present involving the connective tissue sheath about the follicles, most dcrmal hair papillae and the cortex of the hair within a few follicles. X 50. B. A higher magnification showing a calcified dermal hair papilla and connective tissue sheath. X 160. 462
463
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FIG. 11. DHT and plucked. Alkaline phosphatase. Numerous alkaline phosphatase positive fibrocytic cells are preseut in the upper dermis and some are located adjacent to the hair follicles. X 50.
immediately associated with the follicles was hyaluronidase-resistant. This material was alcian blue positive at pH 2.5 and negative at pH 0.4. The calcified areas within the follicles
white or plucking. Although there was an increased number of fibrocytic cells in animals treated with iron dextran or egg white injections alouie, these cells as well as fibrocytes of
and the connective tissue sheath were PAS normal skin were alkaline phosphatase negative. positive and diastase-resistant. Specimens tnken The question arises as to the origin of the at 3 and 4 days contained telogen follicles. alkaline phosphatase positive fibrocytic cells. Sections from these specimens showed only small Fames and Barker (8, 9) have studied the areas of calcification within follicles and in- development of alkaline phosphatase activity or bone marrow fleck components and of fibroappreciable calcification of adjacent dermis. Iron blast-like cells developing in marrow cultures. was not found in any of the specimens from ani- On the basis of morphological observations, al-
volving the connective tissue sheath, without
mals given DHT and plucked. These results kaline phosphatase activity, and the absence of indicate that calcification occurs much more non-specific esterase and acid phosphatase activity, Fames and Barker (8, 9) concluded that readily in anagen phase follicles.
the cells were derived from the capillary
COMMENT
One of the more striking and perhaps the most
significant histoehemical finding in this study
was the appearance of alkaline phosphatase positive fibrocytic or fibroblast-like cells prior to tissue calcification. These cells were demon-
endothelium of the marrow fleck. This raises
the question as to whether the cells we observed arc derived from normal strains of fibrocytes present in the dermis, under the influence
of DHT and an elicitor, or whether the cells migrated into the dermis from the capillary
strated to be present prior to or at the time endothelium under these influences. The close of calcification in animals given oral DHT anatomical association of the alkaline phosphaand subsequent injection of iron dextran, egg
tase positive fibrocytic cells with capillary endo-
464
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
thelium, observed in several instances in our material, would support derivation from capillary endothelium. Further histologic and histochemical evaluation of these cells should be carried out. Although alkaline phosphatase has been constantly found in association with zones of calcification in cartilage and bone, its role in the calcifying mechanism remains unsettled. Fleish
increased amount of acid mucopolysaccharides prior to or at the earliest stages of calcification
and the presence of an increased amount of acid mucopolysaccharide after iron dextran or egg white injection alone, would tend to rule out the possibility of the acid mucopolysaccha-
ride appearing secondary to calcification in these particular experimental studies. It is interesting that out histochemical studies showed
and Neuman (10) have demonstrated that a large amount of acid mucopolysaccharidc plasma ultrafiltrate contains one or more sub- or acid mucopolysaccharide-protcin complexes stances which inhibit calcium phosphate nuclea- to be present in the subcutaneous tissue after tion and precipitation. These inhibiting sub- egg white injection and that calcification occurstances presumably prevent mineralization of red at these sites in the subcutaneous tissue normal soft tissue. Neuman and Neuman (11) as well as in sites showing an increased amount have emphasized the fact that normal serum is of hyaluronie acid in the dermis. In some insupersaturated with regards to crystal growth stances however, calcium and/or iron appeared but that even when there is pathological eleva- to be intimately associated with collagen fibers tion of serum calcium, it rarely if ever reaches as well as located between the fibers. There is the level required for precipitation or new crys- considerable evidence (10, 11) that collagen tal formation. Ficish and Neuman (10) have or some product of collagen may at times act found that certain polyphosphates act to in- as a matrix substance for calcification. Histohibit calcium phosphate nucleation and precip- chemical studies of Johnson and Helwig (3) itation, and suggest that the role of alkaline and chemical studies of Mathieson and Fearse phosphatase may be that of inactivation of the (15) have demonstrated that extracellular inpolyphosphate inhibitors. The early appear- terfibrillar ground substance acid mucopolyance of the alkaline phosphatase active fibro- saccharide is predonnnantly, if not entirely, cytic cells and the observation that the cells hyaluronic acid. Chondroitin sulfate found on were not always in the immediate vicinity of chemical analysis of whole skin is probably the initial calcifying sites, would support this intimately associated with collagen fibers (3, 16, concept. The fact that local tissue changes are 17) and may play a role in the binding of of major importance in this type of calcinosis, iron and/or calcium to these fibers. once the changes are induced, is demonstrated Our findings support those of Selye (1) by the findings of Selye, Gabbiani, and Tuch- that mast cells are increased in tissue near weber (12) that DHT sensitized rat skin trans- calcified sites. We also found an increased planted to normal recipients 17 hours after number of mast cells in rat skin treated with iron dextran injection, proceeded to calcify.
iron dextran or egg white injection alone. Selye,
The possible importance of acid mucopoly- Dieudonnc, and Veilleux (18) reported the saccharides (13, 14) and/or collagen (10, 11) calcification of discharged mast cell granules. to act as a template or matrix substance for This occurred after subcutaneous injections of
initial crystal formation has been reported. minute amounts of mast cell depleters after Johnson, Graham, and Hclwig (2) reported the sensitization with oral DHT and intravenous finding of acid mucopolysaccharides at sites of cutaneous calcification in humans and suggested
iron dextran. We did not observe calcification of discharged mast cell granules in this study, that tissue injury of some type may lead to however the experimental sensitization proceelaboration of acid mucopolysaccharidc, which dure was different and mast cell depleters were
then plays a role in the binding of calcium. not used. Since mast cell granules contain The possibility that the acid mucopolysaccha- heparin, a sulfated acid mueopolysaccharide,
ride occurred secondary to the calcification it would not be unexpected that it could act could not be ruled out since the lesions studied as a matrix substance to bind calcium or iron represented a late stage with well developed in cxtraeellular locations. calcioosis. The finding in this study of an The exact mechanisms of tissue calcification
EXPERIMENTAL CUTANEOUS CALCINOSIS
465
remain unknown, however one can speculate dermis to the subcutaneous tissue. Iron was as to some of the processes occurring in this contained in fibrocytic cells but not within particular type of cutaneous calcinosis experi- mast cells. In animals given DHT and iron mentally induced in rats. D11T appears to dextran, egg white, or plucked, initial calcifisensitize capillary endothelium or preexisting cation usually occurred within 24 to 48 hours. dermal fibrocytes so that certain types of trauma Ailcalinc phosphatase positive fibroblast-likc or injury cause proliferation of alkaline phos- cells appeared prior to initial calcification phatase positive fibrocytic or fibroblast-like but were not always located immediately adcells. The liberation of alkaline phosphatase, and jacent to initially calcified sites. These cells perhaps other substances from these cells may were not seen in sections from control animals act by destruction of inhibitors normally present, or in normal skin. The origin of the alkaline such as polyphosphates. Initial calcium de- phosphatase positive cells was not definitely posits occur at sites of matrix substances. determined but may be from capillary endotheHyaluronic acid in extracellular spaces, collagen hum or preexisting dermal fibrocytes. Alkaline fibers (perhaps containing chondroitin sulfate), phosphatase may act by destroying polyphosand/or hcparin from mast cell granules may phatc inhibitors normally present in the tissues.
Initial sites of calcification appeared to be act as matrix substances. The results of this preliminary histologic, and related to areas rich in acid mucopolysaccharides histochemical study suggests a need for addi- or in some instances, collagen fibers. Hyaluronic tional investigations including the study of a acid in cxtraccllular spaces, collagen fibers larger number of specimens, the nse of various (perhaps containing chondroitin sulfate B) sensitizers and clicitors, the effect of local and and/or acid mucopolysaccharides associated systemic hormones, and the study of additional with hair follicles appeared to act as matrix enzyme systems suspected of playing a role substances. in tissue calcification. sUMMARY
Cutaneous calcinosis was induced in rats by sensitization with oral dihydrotrachystcrol (DHT) and the use of subcutaneous iron dcx-
tran, subcutaneous egg white, or plucking (manual depilation). Controls consisted of ani-
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E. B.: Histochemistry of the acid muco-
polysaccharides in cutaneous calcification. J. Invest. Dorm., 42: 215, 1964. 3. JOHNSON, W. C. AND HELwm, E. B.: Histo-
chemistry of the cutaneous interfibrillar
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alone, egg white alone and normal skin. Tissue
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specimens were taken at intervals between 18 hours to 6 days after treatment, and were prepared by appropriate methods to demonstrate calcium, iron, mast cells, various acid mucopolysaccharidcs, and alkaline phosphatase. The DHT treated controls did not show signifi-
cant abnormal change except for a possible increase in the number of fibrocytes and mast
cells. Iron dcxtran and egg white treated controls showed a definite increase in the number of dermal fibrocytes and mast cells, a moderate number of inflammatory cells in the
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5. Hoomiwimon, C. J. M. AND SMIT5, C.: The specificity of the periodic acid-Schiff tech-
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6. GRAHAM J. H., JoHNsoN W. C., BUR000N, C. F., JR. AN0 HELwJG, E. B.: Tinea capitis:
a histopathological and histochemical study. Arch. Derm. (Chicago), 89: 528, 1964.
7. Anm-Hu, S. AND HINEHART, J.: Fuchsinaldehyde staining of sulfated mucopolysaccharides and related substances. J. Nat. Cancer last., 13: 232, 1952.
S. FAENRS, P. AND BARKER, B. E.: Cytochemical
studies of human bone marrow fibroblast-
subcutaneous tissue but rarely extending into like cells: 1. alkaline phosphatase. Exp. Cell Res., 29: 278, 1963. the dermis, and increased amounts of material P. AND BARKER, B. E.: Cytochemical with histochcmical properties of hyaluronic 9. FARNE5, studies of human bone marrow fibroblastacid located in the extracellular intcrfibrillar like cells. Amer. J. Path., 44: 481, 1964. ground substance. Sections from rats following 10. FLRISH, H. AND NEUMAN, W. F.: Mechanisms of calcification: role of collagen, polyphosiron dextran injections showed intracellular phates, and phosphatase. Amer. J. Physiol., 200: 1296, 1961. and extracellular iron extending from the mid-
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THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
11. NEUMAN,
W. F. AND NEUMAN, M. W.: The
Chemical Dynamics of Bone Mineral, 209 pp.
Chicago, Ill., University of Chicago Press. 1958.
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rat skin. Canad. J. Biochem. Physiol.,
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12. SELvE, H., GAEEIANI, C. AND TUCHWEBER,
B.: Calciphylaxis: Passive transfer. Science, 143:
15. MATHIEsON, J. M. AND PEARcE, R. H.: The glycosaminoglycan of the ground substance
365, 1964.
P., LINKER, A. AND METER, K.: The acid mucopolysaccharidcs of connective
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tissue. II. Further experiments on chon-
13. LEvINE, M. D., llumN, P. S., FOLLIS, R. H., JE. AND HOWARD, J. E.: Histochemical
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droitin sulfate B. Arch. Biochem., 69: 435,
studies oo calcinosis universalis with respect 17. BANOA, I. AND BALO, J.: Isolation of neutral to possible relationship betweeD normal and heteropolysaccharide containing mucopropathologic calcification. In mctabDlic intertein from bovine Achilles tendon with the relationships, Transactions of First Conaid of collagenmucoproteinase. Biochem. J., 74: 388, 1960. ference, p. 41. New York, Josiah Macy, Jr. 18. SELTE, H., DIRUDONNE, J. M. AND VEILLEUX, Foundation, 1949.
R., REvEN, C. AND SAWyER, J.: The of dye binding on experimental
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71: 511, 1954.
Sidercalciphylactic sensitization to
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linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
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skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-
No warranty is given about the accuracy of the copy.
Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.
Cell Biol., 27: 603, 1965.
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.
30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-
lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.
32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.
34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:
Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.
44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.
45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.
46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,
Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by
Res., 33: 176, 1964.
Tissue calcification is a dynamic process and the end stage, when studied by morphological, histochemical, or chemical methods, yields only
was fixed in neutral buffered formalin for paraffin-
limited information concerning the pathogenesis.
hours, and at 3, 4, 5, and 6 days after injection. The number of animals and the time period of obtaining tissue after treatment with DHT and egg white were as follows: 18 hours, 2; 24 hours, 3; 32 hours, 2; 40 hours, 1; 48 hours, 2; 56 hours, 1; 3 days, 1; 4 days, 1; and 5 days, 1. Specimens
embedding. A total of 34 animals treated with DHT and iron dextran were studied and tissue
Because of the difficulty in obtaining adequate tissue specimens representing documented early phases of pathologic calcinosis in humans, it has been necessary to study experimental cal-
specimens were obtained at 18, 24, 32, 40 and 48
cinosis in laboratory animals. Selye (1) has from animals treated with DHT and plucking were taken at 24 hours, 32 hours, 48 hours, 56 hours,
described excellent methods of inducing experimental cutaneous calcinosis in the rat. Although Selye and others have published detailed biolog-
3 days and 4 days (1 animal used F or each interval).
Controls consisted of normal animals and animals treated with only DHT, iron dextran or egg white.
ical data concerning experimental calcinosis Normal skin from the back of 4 animals was ("calciphylaxis"), adequate histoehemical and studied. Tissue specimens were obtained from 7 animals treated with DHT alone, and these were
histopathological features of early developmental stages have not been described. In this paper we shall report our observations in a preliminary histopathologic and histochemical study of experimental cutaneous calcinosis in the rat.
taken at intervals of 1, 2, 3, and 5 days after
ingestion of DHT. Specimens from animals (1 each interval) treated with iron dextran were taken at 24, 32, 48, and 56 hours, and at 3, 4, and 5
days. Tissue specimens from control animals
treated with egg white were obtained at the same intervals as the iron dextran controls. Formalin-fixed, paraffin-embedded tissue secMATERIAL5 AND METHODS tions were prepared by the following methods: The animals used were female Lewis albino hematoxylin and eosin; the colloidal iron technic, rats, each weighing approximately 100 grams, ex- with and without bovine testicular hyaluronidnse cept for 1 group of female Wistar rats weighing digestion for 1 hour at 37 C; the alcian blue stain approximately 200 grams. Oral dihydrotachysterol at pH 2.5 and 0.4; the aldehyde-fuchsin stain at (DHT), 1 mg in 0.5 ml corn oil, was used as the pH 1.7 and 0.4; periodic acid-Schiff (PAS) reacsensitizer, and an elicitor was given 24 hours later. tion, with and without diastase digestion; potasElicitors used were iron dextran, egg white and sium ferrocyanide and hydrochloric acid method plucking. Two ml of a 17% solution of iron dcx- for iron; and the alizarin red S and the von Kossa tran in distilled water, or 5 ml of a 50% solution methods for calcium. The technics followed and of egg white in distilled water, was inj ected the basis for our interpretation of a positive reacsubcutaneously in the midline of the back. The tion, except for the von Kossa stain, have been animals were lightly anesthetized with ether at the reported (2, 3). The von Kossa stain (4) is based time of injection or plucking (mechanical dep- on the combination of silver with anion salt which ilation). Tissue specimens taken from the midline may be carbonate, phosphate, oxalate, sulfate, of tbe back were immediately frozen with dry chloride, or sulfocyanide, and reduction to meice and used for cryostat-sectioning. Adjacent skin tallic silver by exposure to light. Those salts remaining in tissue sections after routine processing
* From
the Skin and Cancer Hospital of Phila- are almost always due to calcium. Urates and uric acids are the only serious source of error and these
delphia, Department of Dermatology, Temple
compounds can be removed by their solubility
University School of Medicine, Philadelphia, Pa.
in lithium carbonate solution. Procedures to demThis investigation was supported in part by re- onstrate calcium, iron, and acid mucopolysacsearch training grant no. 2A-5289 (C2), from the charides were then performed. Non-specific alkaline 19107.
National Institute of Arthritis and Metabolic
Diseases, and grant no. CA 07478, from the Na- phosphatase preparations using acetone-fixed, cryotional Cancer Institute, U. S. Public Health Serv- stat-sections were performed using naphthol ASMX phosphate (Sigma) and fast blue RH or red ice, Bethesda, Maryland 20014. Presented at the Twenty-fifth Annual Meeting violet LB salts as substrate and coupling dyes of The Society for Investigative Dermatology, respectively. Sections mounted on cover slips, or free sections, were incubated 10 to 15 minutes in Inc., San Francisco, Calif., June 22, 1964. 453
454
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
the substrate at pH 8.6 and some were counterstained with toluidine blue, aldehyde-fuehsiu, or
These reactions arc consistent with a sulfated acid mucopolysaccharide such as heparin. Alalizarin red S. Sections were mounted in glycerolgelatin or were dehydrated and passed quickly though purified heparin has been reported to through xylene to Permount. Occasional cryostat- be PAS negative, (5) protein complexes or sections were stained with alizarin red S to rule other material present in the granules may out the possibility of the calcium being leeched give rise to the positive PAS reaction. Extraout by formalin-fixation or paraffin-embedding. cellular material located in the dermal anagen Acid mucopolysaccharides and mucopolysaccharide-protein complexes were identified by the hair papillac was colloidal iron positive, hyalucolloidal iron, alcian blue, aldehyde-fuchsin, and ronidase resistant, alcian blue positive at pH PAS techuics. Mast cells were most readily identi- 2.5 and 0.4, aldehydc-fucbsin positive at pH fied by the aldehyde-fuchsin stain but were also 1.7 and 0.4, and PAS negative. These reactions demonstrated by alcian blue, colloidal iron, and indicate a sulfated acid mucopolysaccharidc and PAS methods. Elastic fibers were demonstrated by the aldehyde-fucbsin stain. Iron was identified by would be consistent with those of chondroitin the ferrocyanide iron stain and calcium was dem- sulfate B. The outer root sheath of the anagen onstrated by alizarin red S and von Kossa methods. hair contained a small amount of reacting Combination methods used to demonstrate multi- material which was colloidal iron positive, alple elements in the same tissue section included methods for: iron, mast cells, and calcium; calcium cian blue positive at pH 2.5 and negative at and alkaline phosphatase and alkaline phosphatase 0.4, aldehydc-fuchsin negative, and PAS negative
and mast cells (demonstrated by toluidine blue or aldehyde-fuchsin). RESULTS AND INTERPRETATION
Normal Skin: Only those histological and histochemical features pertinent to this study will be described. Prominent differences of rat skin from that of human include the relative thinness of the epidermis, absence of sweat glands, a well developed panniculus carnosus muscle (PCM), a layer of fat cells above the PCM and the alterations associated with the hair cycle. The upper third of the dermis is composed chiefly of collagen fibers aud is asso-
after diastase digestion. Most of this material was hyaluronidasc-resistant. Due to the small amount of material, histochemical characterization was not entirely satisfactory. Graham et al.
(6) have demonstrated the presence of hyaluronic acid in the outer root sheath of human hair follicles. The cortex of the hair, especially in the middle and lower portions, reacted to the aldehydc-fuchsin stain at pH 1.7 and to a lesser
extent at pH 0.4. Since other stains for acid mucopolysaccharides were negative or only weakly positive in the hair cortex, the nature of the aldehyde-fuchsin reactive material is unknown. Abul-Haj and Rinehart (7) suggested
ciated with a relatively greater number of that aldehyde-fuchsin has an affinity for organic
fibrocytes than that seen in human skin. The middle third, in the anagen phase, is composed
sulfur containing compounds in addition to sulfated acid mucopolysaccharidcs. The high sul-
of fat cells with some collagen fibers inter- fur content of hair may be the basis for the
spersed between the fat cells, and contains the affinity observed. Only a small amount of acid middle aud lower portions of the hair follicle. mucopolysaccharidc could be demonstrated in This zone is greatly diminished in the telogen the extracellular interfibrillar ground substance.
phase, and the resting follicle is contained
Most of tlus material was located between
primarily within the collagen of the upper derthe fat cells and about the hair follicles in the mis. The lower third of the dermis is composed of the PCM and a narrow zone of collagen zunc above the PCM. This extracellular interfibers beneath the PCM, separating it from the fibrillar material showed reactions indicating hysubcutaneous tissue. Mast cells were located ad- aluronic acid, i.e. colloidal iron positive, hyjacerit to blood vessels at all levels in the dermis aluronidase labile, alcian blue positive at pH
and the number, even at concentrated sites, 2.5 and negative at pH 0.4, aldehydc-fucbsin rarely exceeded ten per high power field. Mast negative, and PAS negative. The aldehyde-
fuchsin stain demonstrated elastic fibers in the stain at pH 1.7 and 0.4, the alcian blue stain upper corium, an absence of elastic fibers in at pH 2.5 and 0.4, the colloidal iron technic, the mid-dermis, a few fibers immediately above and the PAS method. Hyaluronidase digestion the PCM and a narrow dense band of fibers did not remove the colloidal iron reactivity. immediately beneath the PCM. Stains for iron cell granules reacted with the aldchyde-fuchsin
455
3
-
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----
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-
EXPERIMENTAL CUTANEOUS CALCINOSIS
FIG. 1. Normal skin. Alkaline phosphatase reaction. A. Capillaries, sebaeeous gland cells, dermal
hair papillae, and a zone about the lower portion of the follicles give a positive reaction. X 50. B. Intense
alkaline phosphatase activity is present in the dermal papilla of an anngen follicle and along a narrow zone extending about the outer root sheath in the lower part of the follicle. X 66.
and calcium were negative. Alkaline phospha- traeellular and intracellular in the zone above tase activity was observed in the capillary- the PCM, an occasional area between the muscle endothelium and sebaeeous gland cells (Fig. 1A), bundles, and a large amount both extraeellular and intense activity in the dermal hair papillae and intracellular beneath the PCM. Technies with a narrow zone extending about the outer showing both iron and mast cells in the same root sheath in the lower portion of the follicle tissue section revealed the mast cells did not (Fig. lB). No alkaline phosphataso activity was contain iron. The PAS technic showed a brownish-red reaction at sites of intracellular demonstrated in mast cells or fibroeytes. DHT Treated: Except for the number of and extraeellular iron. The reactivity presumafibroeytes and mast cells, no histiologie or histo- bly is due to dextran, although the possibility of chemical changes from that seen in normal skin recombination of iron with other tissue elements was observed in specimens taken 1 to 5 days must be considered. A few to a moderate numafter administration of DUT. An increased num- ber of polymorphonuclear leukocytes, and small ber of mast cells and fibrocytes was observed in round cells, as well as increased numbers of the latter part of the peroid, however, due to fibrocytic and histiocytic cells were present the limited number of specimens studied, this in the dermis and subcutaneous tissue beneath apparent moderate increase in number could the PCM. Specimens taken at 32 hours showed a relatively larger proportion of the iron to be be a chance occurrence.
Iron Dextran Treated: Specimens taken at located intraeellularly. An additional finding was 24 hours showed an increased number of fibro- the presence of an increased amount of extraeytcs and mast cells located principally in the cellular interfibrillar material showing histomiddle of the dermis and beneath the PCM. chemical properties of hyaluronie acid, located Iron was demonstrated to be present within a principally in the middle of the dermis between few fibrocytes in the upper corium, both cx- fat cells and about hair follicles, and beneath
456
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
the PCM. Specimens taken at later intervals
reactions indicate that the material is a non-
showed an increased amount of hyaluronic acid sulfated acid mucopoiysaccharide or acid mucoin the interfibrillar ground substance, and a per- polysaceharide-protein complex and does not sistence of the increased numbers of fibrocytic contain a significant quantity of hyaluronic
acid. Specimens taken at 32 and 48 hours
cells.
Egg White Treated: Specimens taken at 24 showed increased numbers of fibrocytes and hours showed a moderate increase in the num- histiocytes, an increased amount of hyaluronic ber of fibrocytes and mast cells in the middle acid and decreased numbers of inflammatory of the dermis and beneath the PCM. An in- cells beneath the PCM. Only an occasional increased amount of extracellular interfibrillar material showing properties of hyaluronic acid was also present in these areas. A moderate number
flammatory cell was observed above the PCM at
any stage. Specimens taken at 3 and 4 days
continued to show an increased number of mast of eosinophilic polymorphonuclear leukocytes cells and increased amounts of hyaluronic acid and small round cells, in addition to increased located principally in the mid dermis and below numbers of histocytie and fibrocytic cells, were the PCM. The number of fibrocytes was somepresent in the subcutaneous tissue and in the what less in tissue taken on the fourth day. dermis beneath the PCM. Some specimens DHT and Iron Dextran Treated: Specimens taken at 18 hours after injection showed some showed a foreign material (egg white) separating the tissue elements in the subcutaneous tissue. increase in the number of fibrocytes and an This material was colloidal iron psoitive, hy- occasional polymorphonuclear leukocyte in the aluronidase-resistant, alcian blue positive at dermis above the PCM. A moderate number of pH 2.5 and negative at 0.4, aldehyde-fuchsin polymorphonuclear leukocytes, mostly eosinopositive at pH 1.7, PAS positive and diastase phils, and an increased number of fibrocytic, resistant (Fig. 2). Egg white smeared on glass histiocytic, and mononuclear cells were present slides showed identical staining reactions. These in the subcutaneous tissue and in the dermis
-
-
--
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---
k$2*4: I', I-.,. -.-e
-:
--
a;jnfe:
-te -—-2,-r--' -& -
-
—-r
-
-
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-
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-
Fia. 2. Egg white treated. PAS with diastase digestion. A foreign material (egg white) separates and replaces normal tissue elements in the subcutaneous tissue and shows intense PAS reactivity. X 19.
457
EXPERIMENTAL CUTANEOUS CALCINOSIS
beneath the PCM. A relatively large amount of nantly in the zone above the PCM, was demintracellular and extracellular iron was present onstrated. Alkaline phosphatase activity was in the zone above the PCM, occasional iron- observed in fibrocytic cells above as well as becontaining fibrocytic cells were located between low the PCM (Fig. 3). The von Kossa and the muscle bundles, and a moderate amount of alizarin red S technics revealed early caliron was present in the dermis and subcutaneous cification, in 1 specimen, above the PCM in tissue beneath the PCM. Tcchnics for acid muco- the same zone which showed an increased polysaccharides showed an increased amount of amount of hyaluronic acid. Most tissue specihyaluronic acid beneath the PCM in the der- mens taken at 48 hours showed patchy areas of mis and subcutaneous tissue. Alkaline phos- calcification in the mid dermis and in the dermis phatase activity was observed in fibrocytic cells beneath the PCM (Fig. 4). Some specimens located in the dermis beneath the PCM. The showed calcification extending into the upper predominant activity was in the cytoplasm, corium almost to the epidermis. Small patchy although the possibility of activity in the nucleus areas of calcification were occasionally seen could not be completely excluded. The cells between the bundles of the IPCM. A moderate initially appeared at sites near capillaries. Tis- number of fibrocytes containing intracellular sue sections prepared to demonstrate both al- iron, as well as extraccllular iron was present.
Much of the extraccllular iron appeared to be coated on or located within the collagen fibers. Mast cells were decreased in number or absent in calcified areas, but were increased in number between and about the calcified sites. Calcifica-
tion of mast cell granules was not observed in sections prepared for demonstration of both calcium and mast cell granules. An increased
44
f
9'
)
4f
kaline phosphatase activity and mast cells revealed that the mast cells did not show alkaline phosphatase activity. Specimens taken at 24 hours showed a greater number of fibrocytes and mast cells above the PCM and a smaller number of polymorphonuclear leukocytcs below the PCM than at 18 hours. An increased amount of hyaluronic acid located predomi-
FIG. 3. DHT and iron dcxtraa treated. Alkaline phosphatase reaction. A moderate number of alkaline phosphatasc positive fibrocytic cells arc located above the PCM and a few arc beneath the PCM at 24 hours after injection. )< 55.
458
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
amount of hyaluronic acid in the mid-dermis and
the subcutaneous tissue to the epidermis. Speci-
beneath the PCM was demonstrated by tech- mens taken at 5 and 6 days showed diffuse flies for acid mucopolysaecharides. Some speci- patchy calcified areas throughout the dermis mens showed calcification of areas in the mid- except for some uninvolved areas in the PCM. dermis without calcification in the lower der- Cells were few to absent in the calcified areas. mis beneath the PCM. A large number of Iron remained limited to the middle and lower fibrocytic cells showing alkaline phosphatase dermis and was not present in calcified areas activity were demonstrated in most specimens. in the upper dermis. Some iron appeared coated Alkaline phosphatase positive cells usually were on or absorbed by the collagen fibers whereas not immediately associated with the calcified some remained between the collagen fibers or areas. Specimens taken at 3 and 4 days showed within cells. Alkaline phosphatase positive fibrosimilar features except the calcified areas occu- eytic cells were numerous in the peripheral pied almost the entire dermis in a diffuse but zones of calcification and in adjacent unealeipatchy involvement including a few calcified fled tissue. Reactions for acid mueopolysaeehaareas within the PCM. Alkaline phosphatase rides revealed an increased amount of hyaluronie positive fibrocytie cells were numerous in the acid in most calcified areas. Calcified areas
upper eorium and activity was not entirely did not show a greater PAS reactivity than intracellular (Fig. 5). The extracellular fine the adjacent collagen. A few sections showed granules may have represented a diffusion artifact. The morphology of the collagen fibers in the calcified areas was greatly altered and many small basophilie granules were present in the hematoxylin- and eosin-stained sections. Iron usually remained localized to the mid-dermis even in those areas where the calcium extended from
small areas of necrosis in the superficial dermis with an associated collection of polymorphonuclear leukoeytes.
DHT and Egg White Treated: Specimens taken at 18 hours showed a moderate increase in the number of fibroeytes and histioeytes in the zone above the PCM. Numerous infiam-
Fm. 4. DIIT and iron dextran treated. Alizarin red S stain for calcium. Most tissue specimens taken at 48 hours showed ealcinm deposition in the mid-dermis and in the dermis beneath the PCM. X 50.
459
EXPERIMENTAL CUTANEOUS CALCINOSIS
.•(.
FIG. 5. DHT and iron dextran treated. Alkaline phosphatase reaction. At 4 days numerous alkaline phosphatase positive fibrocytic cells were present in the upper dermis. Some activity appeared to be extraeellular, however this might be a diffusion artifact. X 81.
matory cells including eosinophilie polymorpho- present in the upper dermis. Mast cells and
nuclear leukoeytes, small round cells, and an increased number of histiocytes and fibroeytes were present in the dermis below the PCM and in the subcutaneous tissue. Fereign material (egg white) separated normal elements of the
hyaluronie acid were present in increased amounts in the mid dermis and beneath the
PCM. One specimen taken at 48 hours showed calcification limited to the zone above the PCM (Fig. 6). The calcified area showed a marked subcutaneous tissue and showed identical bisto- increase of hyaluronie acid, whereas there was chemical characteristics as that of the material a relatively small amount of hyaluronie acid seen in animals injected with egg white without present below the 2CM (Fig. 7, A and B). DHT treatment. An increased amount of hyalu- Alkaline phosphatase positive fibrocytie cells ronie acid was demonstrated in the zone above were numerous below the PCM and only a the PCM, and a lesser amount between bun- few were present in the calcified area (Fig. 8). dles of the PCM and in the subjacent der- Another specimen taken at 48 hours showed mis. A few to moderate number of alkahne large areas of calcification in the subcutaneous pliosphatase positive fibroeytie cells were present tissue and only small areas of early calcification in the dermis beneath the 2CM. Specimens above the PCM between the fat cells. Teehnies taken at 24 hours showed similar changes for acid mueopolysaeeharides were positive in except that alkaline phosphatase positive cells all areas of calcification. Specimens taken at 3 were present in the mid-dermis as well as be- and 4 days demonstrated extension of calcified neath the 2CM. The number of acute inflam- areas throughout the dermis with the 2CM matory cells in most specimens was less than being least affected. In densely calcified areas, that at 18 hours. Specimens taken at 32 hours colloidal iron reactive material was only partially showed similar features except the number of hyaluronidase-labile. Specimens taken at all alkaline phosphatase positive cells had increased intervals were negative for iron. The connecin the middle and lower dermis and a few tive tissue sheath about hair follicles were cal-
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EXPERIMENTAL CUTANEOUS CALCINOSIS
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cified in the later stages but the dermal papillae positive material in the outer root sheath, an and follicular epithelium usually were not calci- increased number of mast cells and fibrocytie fied.
cells, and a few to a moderate number of
DHT Treated and Plucked: Specimens taken alkaline phosphatase positive fibrocytic cells in at 24 hours showed early anagen follicles. The the upper dermis. No sites of calcification were follicles were distorted, especially in the upper observed. The follicles in both specimens were
half, dilated, and contained loose keratinous in the telogen phase. A specimen taken at material. Fibrocytic cells and mast cells were 56 hours showed distorted anagen follicles. The
increased in number in the upper eorium. collagen bundles and hair follicles in the upper Calcium was demonstrated in some follicles half of the dermis showed minute basophilic near the level of the sehaeeous gland. Loose granules in hematoxylin- and eosin-stained seckeratinous material within follicles and the con- tions. A dense zone of calcification involved the nective tissue sheaths about the follicles were connective tissue sheath about the follicles, most
the predominant anatomical structures calci- dermal hair papillae, and the cortex of the fied. Alkaline phosphatase positive fibrocytie hair within some follicles (Fig. 10, A and B). cells were present in the upper corium and A patchy diffuse calcification was observed in some showed a close relationship to capillaries the upper half of the corium in most areas. adjacent to involved follicles (Fig. 9). Increased Numerous alkaline phosphatase positive fibroamounts of hyaluronie acid were present in the cytie cells were present in the upper eorium mid-dermis. Most of the colloidal iron posi- and in some areas were found in close proxtive material associated with the follicular epi- imity to the hair follicles (Fig. 11). An increased thelium was hyaluronidase-resistant. Specimens amount of interfibrillar ground substance matetaken at 32 and 48 hours showed some distortion rial in the mid-dermis and a lesser amount bein the upper part of the follicles, an increased tween the follicles in the upper dermis showed amount of hyaluronie acid in the mid-dermis, histochemical properties of hyaluronie acid. moderately increased amounts of colloidal iron Most of the colloidal iron positive material
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Fin. 10. DHT and plucked. Von Kossa stain for calcium. A. At 56 hours a dense zone of calcification was present involving the connective tissue sheath about the follicles, most dcrmal hair papillae and the cortex of the hair within a few follicles. X 50. B. A higher magnification showing a calcified dermal hair papilla and connective tissue sheath. X 160. 462
463
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EXPERIMENTAL CUTANEOUS CALCINOSIS
FIG. 11. DHT and plucked. Alkaline phosphatase. Numerous alkaline phosphatase positive fibrocytic cells are preseut in the upper dermis and some are located adjacent to the hair follicles. X 50.
immediately associated with the follicles was hyaluronidase-resistant. This material was alcian blue positive at pH 2.5 and negative at pH 0.4. The calcified areas within the follicles
white or plucking. Although there was an increased number of fibrocytic cells in animals treated with iron dextran or egg white injections alouie, these cells as well as fibrocytes of
and the connective tissue sheath were PAS normal skin were alkaline phosphatase negative. positive and diastase-resistant. Specimens tnken The question arises as to the origin of the at 3 and 4 days contained telogen follicles. alkaline phosphatase positive fibrocytic cells. Sections from these specimens showed only small Fames and Barker (8, 9) have studied the areas of calcification within follicles and in- development of alkaline phosphatase activity or bone marrow fleck components and of fibroappreciable calcification of adjacent dermis. Iron blast-like cells developing in marrow cultures. was not found in any of the specimens from ani- On the basis of morphological observations, al-
volving the connective tissue sheath, without
mals given DHT and plucked. These results kaline phosphatase activity, and the absence of indicate that calcification occurs much more non-specific esterase and acid phosphatase activity, Fames and Barker (8, 9) concluded that readily in anagen phase follicles.
the cells were derived from the capillary
COMMENT
One of the more striking and perhaps the most
significant histoehemical finding in this study
was the appearance of alkaline phosphatase positive fibrocytic or fibroblast-like cells prior to tissue calcification. These cells were demon-
endothelium of the marrow fleck. This raises
the question as to whether the cells we observed arc derived from normal strains of fibrocytes present in the dermis, under the influence
of DHT and an elicitor, or whether the cells migrated into the dermis from the capillary
strated to be present prior to or at the time endothelium under these influences. The close of calcification in animals given oral DHT anatomical association of the alkaline phosphaand subsequent injection of iron dextran, egg
tase positive fibrocytic cells with capillary endo-
464
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
thelium, observed in several instances in our material, would support derivation from capillary endothelium. Further histologic and histochemical evaluation of these cells should be carried out. Although alkaline phosphatase has been constantly found in association with zones of calcification in cartilage and bone, its role in the calcifying mechanism remains unsettled. Fleish
increased amount of acid mucopolysaccharides prior to or at the earliest stages of calcification
and the presence of an increased amount of acid mucopolysaccharide after iron dextran or egg white injection alone, would tend to rule out the possibility of the acid mucopolysaccha-
ride appearing secondary to calcification in these particular experimental studies. It is interesting that out histochemical studies showed
and Neuman (10) have demonstrated that a large amount of acid mucopolysaccharidc plasma ultrafiltrate contains one or more sub- or acid mucopolysaccharide-protcin complexes stances which inhibit calcium phosphate nuclea- to be present in the subcutaneous tissue after tion and precipitation. These inhibiting sub- egg white injection and that calcification occurstances presumably prevent mineralization of red at these sites in the subcutaneous tissue normal soft tissue. Neuman and Neuman (11) as well as in sites showing an increased amount have emphasized the fact that normal serum is of hyaluronie acid in the dermis. In some insupersaturated with regards to crystal growth stances however, calcium and/or iron appeared but that even when there is pathological eleva- to be intimately associated with collagen fibers tion of serum calcium, it rarely if ever reaches as well as located between the fibers. There is the level required for precipitation or new crys- considerable evidence (10, 11) that collagen tal formation. Ficish and Neuman (10) have or some product of collagen may at times act found that certain polyphosphates act to in- as a matrix substance for calcification. Histohibit calcium phosphate nucleation and precip- chemical studies of Johnson and Helwig (3) itation, and suggest that the role of alkaline and chemical studies of Mathieson and Fearse phosphatase may be that of inactivation of the (15) have demonstrated that extracellular inpolyphosphate inhibitors. The early appear- terfibrillar ground substance acid mucopolyance of the alkaline phosphatase active fibro- saccharide is predonnnantly, if not entirely, cytic cells and the observation that the cells hyaluronic acid. Chondroitin sulfate found on were not always in the immediate vicinity of chemical analysis of whole skin is probably the initial calcifying sites, would support this intimately associated with collagen fibers (3, 16, concept. The fact that local tissue changes are 17) and may play a role in the binding of of major importance in this type of calcinosis, iron and/or calcium to these fibers. once the changes are induced, is demonstrated Our findings support those of Selye (1) by the findings of Selye, Gabbiani, and Tuch- that mast cells are increased in tissue near weber (12) that DHT sensitized rat skin trans- calcified sites. We also found an increased planted to normal recipients 17 hours after number of mast cells in rat skin treated with iron dextran injection, proceeded to calcify.
iron dextran or egg white injection alone. Selye,
The possible importance of acid mucopoly- Dieudonnc, and Veilleux (18) reported the saccharides (13, 14) and/or collagen (10, 11) calcification of discharged mast cell granules. to act as a template or matrix substance for This occurred after subcutaneous injections of
initial crystal formation has been reported. minute amounts of mast cell depleters after Johnson, Graham, and Hclwig (2) reported the sensitization with oral DHT and intravenous finding of acid mucopolysaccharides at sites of cutaneous calcification in humans and suggested
iron dextran. We did not observe calcification of discharged mast cell granules in this study, that tissue injury of some type may lead to however the experimental sensitization proceelaboration of acid mucopolysaccharidc, which dure was different and mast cell depleters were
then plays a role in the binding of calcium. not used. Since mast cell granules contain The possibility that the acid mucopolysaccha- heparin, a sulfated acid mueopolysaccharide,
ride occurred secondary to the calcification it would not be unexpected that it could act could not be ruled out since the lesions studied as a matrix substance to bind calcium or iron represented a late stage with well developed in cxtraeellular locations. calcioosis. The finding in this study of an The exact mechanisms of tissue calcification
EXPERIMENTAL CUTANEOUS CALCINOSIS
465
remain unknown, however one can speculate dermis to the subcutaneous tissue. Iron was as to some of the processes occurring in this contained in fibrocytic cells but not within particular type of cutaneous calcinosis experi- mast cells. In animals given DHT and iron mentally induced in rats. D11T appears to dextran, egg white, or plucked, initial calcifisensitize capillary endothelium or preexisting cation usually occurred within 24 to 48 hours. dermal fibrocytes so that certain types of trauma Ailcalinc phosphatase positive fibroblast-likc or injury cause proliferation of alkaline phos- cells appeared prior to initial calcification phatase positive fibrocytic or fibroblast-like but were not always located immediately adcells. The liberation of alkaline phosphatase, and jacent to initially calcified sites. These cells perhaps other substances from these cells may were not seen in sections from control animals act by destruction of inhibitors normally present, or in normal skin. The origin of the alkaline such as polyphosphates. Initial calcium de- phosphatase positive cells was not definitely posits occur at sites of matrix substances. determined but may be from capillary endotheHyaluronic acid in extracellular spaces, collagen hum or preexisting dermal fibrocytes. Alkaline fibers (perhaps containing chondroitin sulfate), phosphatase may act by destroying polyphosand/or hcparin from mast cell granules may phatc inhibitors normally present in the tissues.
Initial sites of calcification appeared to be act as matrix substances. The results of this preliminary histologic, and related to areas rich in acid mucopolysaccharides histochemical study suggests a need for addi- or in some instances, collagen fibers. Hyaluronic tional investigations including the study of a acid in cxtraccllular spaces, collagen fibers larger number of specimens, the nse of various (perhaps containing chondroitin sulfate B) sensitizers and clicitors, the effect of local and and/or acid mucopolysaccharides associated systemic hormones, and the study of additional with hair follicles appeared to act as matrix enzyme systems suspected of playing a role substances. in tissue calcification. sUMMARY
Cutaneous calcinosis was induced in rats by sensitization with oral dihydrotrachystcrol (DHT) and the use of subcutaneous iron dcx-
tran, subcutaneous egg white, or plucking (manual depilation). Controls consisted of ani-
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