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Proteins of the ground substance of human dermis
S.S. SPICER, R. G. HORN and B. K. WEXZEL
156 41. 42. 43. 44. 45.
H. 1. ZEYA and J. K. SPITZNAGEL,Science 142, 1085 (1963). R. C. SKARNESand D. W. WATSON, J. exp. Med. 104, 829 0956). E. S. GOLUB and J. K. SPITZNAGEL,J. Immun. 95, 1060 (1966). A. JANOFF and B. W. ZWEIFACH,J. exp. Med. 120, 747 (1964). H. I. SALA, H. R. ROaERTS and J. C. HERION,J. olin. hwest. 46, 580(1967). COMMENTS
Dr. Rebuck nEade the point that azurophilic granules are not detected in adult human neutrophils: be wondered if the so-called toxic granules are one of the primary populations that are rapidly formed and therefore might have more primary granules, so that what we have been calling toxic granules may be another granule. Dr. Spicer said that this point--the fact that, after administration of endotoxin, peripheral smears show this toxic granulation of the leukocytes--actually led to the histochemical studies. He thought that the endotoxin caused the release of cells at an earlier stage in their development (compared to normal); therefore, the cilculating leukocytes are relatively early heterophils and so are richer in the primary type granule. He has seen no histochemical difference between the circulating leukocyte with sulfated mucosaccharide-rich granules (presumably, toxic granules) and the bone-marrow early heterophil with abundant granules rich in sulfated mucosaccharide. Dr. Cline commented that uptake of S3~ may not be reliable as an indication of the presence of sulfated mucopolysaccharide because it can be shown that, when sulfur or selenium is added to these cells, sulfur-sulfur or selenium-sulfur bonds form by addition to free--SH groups. He asked if the neutrophil was capable of replenishing its armory of granules after phagocytosis and granule dischai go, because there is no biochemical evidence of regeneration of the granule enzymes. The speaker defended the specificity of the autoradiographic method with S"~'50~- on the basis of correlation with histochemical staining for sulfate in mucosaccha,ide and because the label is removed by brief treatment with acidified methanol. This metholysis is known to eliminate sulfate from sulfated mucopolysaccharide but not to degrade protein or remove cystine or methionine sulfur. He also said that all evidence indicates that the cells cannot regenerate primary granules after the initial production of these has terminated. Once the primary and secondary granules are used tip, they are gone for good. Dr. Gladner also thought that the chances for the S35 labeling to be due to formation of a bond with - - S H of protein were very small. Dr. Pearce's comments on the presumed capillary barrier to the flow of proteins into and out of the extravascular space are reported in expanded form at the end of this section.
Proteins of the ground substance of human dermis THESE studies were undertaken to investigate the composition of the ground substance, the extravascular, extrafibrillar phase of human dermis. The basic question, in view of previous work l, 2 supported by our own evidence3, was this--Is there a capillary barrier to the flow of proteins into and out of the extravascular space, particularly in connective tissues? The specimens, obtained at autopsy of persons who had died in an acute incident, were I ~ by 8-in. pieces of skin excised from the thorax just posterior to the lateral line. Duplicate 4-g samples of dermis from thirteen subjects (six males and seven females, 17-81 yr old) were prepared by removal of subcutaneous tissue, fat, and epidermis. The dermis was minced to a thick paste, lyophilized, extracted with petroleum ether in the cold, and then subjected to exhaustive extraction with isotonic saline. The saline extracts were adjusted to 50 ml/g fresh weight of tissue and were filtered through glass wool. The extracts were rich in nitrogen, and the nitrogen content corresponded well with the protein content (Table 1). When the extract was concentrated and subjected to electrophoresis on cellulose acetate, the distribution of proteins among the fractions resembled very closely that seen in human serum (Table 2). On disc electrophoresis, almost all the major fractions of serum were present. lmmunoelectrophoresis also demonstrated almost all the fractions that can be deteeted in plasma. The volume of plasma equivalent to the proteins present was calculated to be 0-2 ml/g fresh weight of dermis. The contamination of the tissue by its content of blood might be questioned. To resolve this, the extracts were analyzed for hemoglobin by the benzidine reaction. 6 The hemoglobin content was less than 1 per cent of that predicted from its content of plasma protein.
Inflammation at the cellular level--I
157
These extracts contained only one polysaccharide in any quantity--hyaluronic acid. 7 Laurent s and Preston et al. 9 have demonstrated independently that this polysaccharide is capable of excluding plasma proteins from its domain. Could not the transudation of plasma proteins in the inflammatory process be due to a breakdown in hyaluronic acid, permitting the proteins to occupy a domain from TABLE I. ANALYSISOF SALINEEXTRACTOF HUMANDERMIS*
Component Nitrogent Hexosamine Hexuronic acid Hydroxyproline Protein: calculated++ measured§
Mean _ S.D. (/zmole/g fresh wt.) 144 1-85 1.55 2-28
± 37 ~ 0.54 ~= 0"38 ± 0.36
12.6 Ez 3-2 10.9 ± 1'7
* Based on replicate samples of thirteen specimens. t By the method of King. 4 ++Protein = N x 14 ;/. 6-25 in milligrams. § By the method of Lowry et alP; results shown in milligrams. FABLE2. RELATIVECONCENTRATIONSOE PROTEINS IN SALINE EXTRACTSOF HUMANDERMIS Electrophoretic fraction Albumin Alpha Beta Gamma
Protein ( ~ of total) 67.6 14.3 10-9 8.0
± ~: ~ ~
5.0 3.0 2.0 3.0
which they were excluded in the normal state? This would explain the apparent change in capillary permeability.
Department o f Pathology, Faculty of Medicine, University of British Columbia, Canada
1. 2. 3. 4. 5. 6. 7. 8. 9.
R . H . PEARCE
REFERENCES J. H. HUMPHREY,A. NEUBERGERand D. J. PERKINS,Biochem. J. 66, 390 (195,). D. R. COOPER and P. JOHNSON, Biochim. biophys. Acta 26, 317 (1957). P. R. SWEENEY, R. H. PEARCEand H. G. VANCE, Can. J. Biochem. Physiol. 41, 2307 (1963). E. J. KING and [. D. P. WOOTTAN, Micro-Analysis in Medical Biochemistry, 2nd edn, p. 11. Grune & Stratton, New York (1951). O. H. LOWRY, N. J. ROSEBROUGH,A. L. PARR and R. J. RANDALL,J. biol. Chem. 193, 265 (1951) G. E. HANKS, M. CASSELL,R. N. RAY and H. CHAPLIN,JR., J. Lab. Clin. Med. 56, 486 (1960). J. M. MATHrESONand R. H. PEARCE, Can. J. Bioehem. PhysioL 41, 2327 (1963). T. LAURENT, Biochem. J. 93, 106 (1964). B. N. PRESTON, M. DAVIESand A. G. OGSTON, Biochem. J. 96, 449 (1965).
156 41. 42. 43. 44. 45.
H. 1. ZEYA and J. K. SPITZNAGEL,Science 142, 1085 (1963). R. C. SKARNESand D. W. WATSON, J. exp. Med. 104, 829 0956). E. S. GOLUB and J. K. SPITZNAGEL,J. Immun. 95, 1060 (1966). A. JANOFF and B. W. ZWEIFACH,J. exp. Med. 120, 747 (1964). H. I. SALA, H. R. ROaERTS and J. C. HERION,J. olin. hwest. 46, 580(1967). COMMENTS
Dr. Rebuck nEade the point that azurophilic granules are not detected in adult human neutrophils: be wondered if the so-called toxic granules are one of the primary populations that are rapidly formed and therefore might have more primary granules, so that what we have been calling toxic granules may be another granule. Dr. Spicer said that this point--the fact that, after administration of endotoxin, peripheral smears show this toxic granulation of the leukocytes--actually led to the histochemical studies. He thought that the endotoxin caused the release of cells at an earlier stage in their development (compared to normal); therefore, the cilculating leukocytes are relatively early heterophils and so are richer in the primary type granule. He has seen no histochemical difference between the circulating leukocyte with sulfated mucosaccharide-rich granules (presumably, toxic granules) and the bone-marrow early heterophil with abundant granules rich in sulfated mucosaccharide. Dr. Cline commented that uptake of S3~ may not be reliable as an indication of the presence of sulfated mucopolysaccharide because it can be shown that, when sulfur or selenium is added to these cells, sulfur-sulfur or selenium-sulfur bonds form by addition to free--SH groups. He asked if the neutrophil was capable of replenishing its armory of granules after phagocytosis and granule dischai go, because there is no biochemical evidence of regeneration of the granule enzymes. The speaker defended the specificity of the autoradiographic method with S"~'50~- on the basis of correlation with histochemical staining for sulfate in mucosaccha,ide and because the label is removed by brief treatment with acidified methanol. This metholysis is known to eliminate sulfate from sulfated mucopolysaccharide but not to degrade protein or remove cystine or methionine sulfur. He also said that all evidence indicates that the cells cannot regenerate primary granules after the initial production of these has terminated. Once the primary and secondary granules are used tip, they are gone for good. Dr. Gladner also thought that the chances for the S35 labeling to be due to formation of a bond with - - S H of protein were very small. Dr. Pearce's comments on the presumed capillary barrier to the flow of proteins into and out of the extravascular space are reported in expanded form at the end of this section.
Proteins of the ground substance of human dermis THESE studies were undertaken to investigate the composition of the ground substance, the extravascular, extrafibrillar phase of human dermis. The basic question, in view of previous work l, 2 supported by our own evidence3, was this--Is there a capillary barrier to the flow of proteins into and out of the extravascular space, particularly in connective tissues? The specimens, obtained at autopsy of persons who had died in an acute incident, were I ~ by 8-in. pieces of skin excised from the thorax just posterior to the lateral line. Duplicate 4-g samples of dermis from thirteen subjects (six males and seven females, 17-81 yr old) were prepared by removal of subcutaneous tissue, fat, and epidermis. The dermis was minced to a thick paste, lyophilized, extracted with petroleum ether in the cold, and then subjected to exhaustive extraction with isotonic saline. The saline extracts were adjusted to 50 ml/g fresh weight of tissue and were filtered through glass wool. The extracts were rich in nitrogen, and the nitrogen content corresponded well with the protein content (Table 1). When the extract was concentrated and subjected to electrophoresis on cellulose acetate, the distribution of proteins among the fractions resembled very closely that seen in human serum (Table 2). On disc electrophoresis, almost all the major fractions of serum were present. lmmunoelectrophoresis also demonstrated almost all the fractions that can be deteeted in plasma. The volume of plasma equivalent to the proteins present was calculated to be 0-2 ml/g fresh weight of dermis. The contamination of the tissue by its content of blood might be questioned. To resolve this, the extracts were analyzed for hemoglobin by the benzidine reaction. 6 The hemoglobin content was less than 1 per cent of that predicted from its content of plasma protein.
Inflammation at the cellular level--I
157
These extracts contained only one polysaccharide in any quantity--hyaluronic acid. 7 Laurent s and Preston et al. 9 have demonstrated independently that this polysaccharide is capable of excluding plasma proteins from its domain. Could not the transudation of plasma proteins in the inflammatory process be due to a breakdown in hyaluronic acid, permitting the proteins to occupy a domain from TABLE I. ANALYSISOF SALINEEXTRACTOF HUMANDERMIS*
Component Nitrogent Hexosamine Hexuronic acid Hydroxyproline Protein: calculated++ measured§
Mean _ S.D. (/zmole/g fresh wt.) 144 1-85 1.55 2-28
± 37 ~ 0.54 ~= 0"38 ± 0.36
12.6 Ez 3-2 10.9 ± 1'7
* Based on replicate samples of thirteen specimens. t By the method of King. 4 ++Protein = N x 14 ;/. 6-25 in milligrams. § By the method of Lowry et alP; results shown in milligrams. FABLE2. RELATIVECONCENTRATIONSOE PROTEINS IN SALINE EXTRACTSOF HUMANDERMIS Electrophoretic fraction Albumin Alpha Beta Gamma
Protein ( ~ of total) 67.6 14.3 10-9 8.0
± ~: ~ ~
5.0 3.0 2.0 3.0
which they were excluded in the normal state? This would explain the apparent change in capillary permeability.
Department o f Pathology, Faculty of Medicine, University of British Columbia, Canada
1. 2. 3. 4. 5. 6. 7. 8. 9.
R . H . PEARCE
REFERENCES J. H. HUMPHREY,A. NEUBERGERand D. J. PERKINS,Biochem. J. 66, 390 (195,). D. R. COOPER and P. JOHNSON, Biochim. biophys. Acta 26, 317 (1957). P. R. SWEENEY, R. H. PEARCEand H. G. VANCE, Can. J. Biochem. Physiol. 41, 2307 (1963). E. J. KING and [. D. P. WOOTTAN, Micro-Analysis in Medical Biochemistry, 2nd edn, p. 11. Grune & Stratton, New York (1951). O. H. LOWRY, N. J. ROSEBROUGH,A. L. PARR and R. J. RANDALL,J. biol. Chem. 193, 265 (1951) G. E. HANKS, M. CASSELL,R. N. RAY and H. CHAPLIN,JR., J. Lab. Clin. Med. 56, 486 (1960). J. M. MATHrESONand R. H. PEARCE, Can. J. Bioehem. PhysioL 41, 2327 (1963). T. LAURENT, Biochem. J. 93, 106 (1964). B. N. PRESTON, M. DAVIESand A. G. OGSTON, Biochem. J. 96, 449 (1965).