C R Y O I M M U N O G L O B U L I N IgGk W I T H M I C R O T U B U L A R ULTRASTRUCTURE ASSOCIATED WITH PYODERMA GANGRENOSUM J. C. WHITE,B. A. ADAMAND K. S. LAU Faculty of Medicine, University of Malaya, Kuala Lumpur
R. W. HORNE Jolm Innes Institute, Norwich, England
R. M. E. PARKHOUSE National Institute for Medical Research, Mill Hill, London NW7, England
PLATES VI-XI U L C E R A T I OofNthe leg with Raynaud’s phenomenon can mark the presence of paraproteinaemia, including cryoglobulinaemia (Ellis, 1964), with altered blood viscosity and flow (Wells, 1970). Cryoglobulins may form intravascular deposits in the cold with local ischaemia, or complexes may be formed within the vessel wall with associated vasculitis; the cryoproteins may be monoclonal or multicomponent (Moroz and Rose, 1971), and the mixed forms may be immune-complexes (Carter, 1973). Monoclonal cryoglobulins are particularly associated with plasma cell and lympho-reticular proliferative conditions, and crystalline structure has been observed in some (Grey and Kohler, 1973). A particular tubular crystalline structure has been reported in a case of IgGk myeloma (Bogaars et al., 1973). Tubular protein structures appear to be of quite widespread and fundamental natural occurrence (Horne, 1971), and we report studies on a IgGk cryoglobulin with this characteristic, occurring in a patient with pyoderma gangrenosum of the leg but without overt myeloma; vascular luminal obstruction appears to underlie the ulceration. MATERIALS AND METHODS Case report A Chinese man of 64 had painful recurrent ulcers on both calves for 3 y, healing with hyperpigmented scars, but progressing to a large (10 x 3 cm) gangrenous ulcer on the right. On cold exposure both legs became blue and painful, with ankle petechiae. All peripheral pulses were felt and blood pressure was 150/90 mm Hg. Pyoderma gangrenosum was diagnosed. Gelling of the serum occurred at 4°C. The bone marrow was not indicative of meyloma. A benign ulcer was present at the pyloric antrum. Over 6 mth his leg ulcers healed with oral prednisolone and later dapsone, though cold intolerance persisted. At this time collapse of L1 and L5 vertebrae was found, though the rest of the skeleton was normal and three further marrow aspirations showed low plasma Received 17 Sept. 1975; accepted 7 Oct. 1975. 1. PATH.-VOL.
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WHITE, ADAM, LAU, HORNE AND PARKHOUSE
cell incidence. Two months later he was admitted in terminal coma with reappearance of calf ulceration and gangrene of fingers. Necropsy was not obtained. Investigations Histological and cytological methods. Biopsies from ulcer edge and gastrocnemius muscle beneath intact skin were formalin-fixed, paraffin-embedded, and sections stained by haematoxylin and eosin, light green and nuclear fast red, Congo red, Mallory's phosphotungstic acid haematoxylin (PTAH ; before and after fibrinolysin treatment) and Martius-ScarletBlue (MSB) stain. Aspiration and needle-trephine biopsies of iliac crest and sternal bone marrow were stained by May-Grunwald-Giemsa, Prussian-blue and PAS. Serum protein investigation. Cellulose acetate electrophoresis was performed in barbitone buffer, pH 8.6; the cryoprecipitate and supernatant separated by centrifugation at 4°C were run separately after melting at room temperature. Relative viscosity of whole serum was determined in a BS/U/M2 U-tube Ostwald microviscometer a t 29", 20" and 3°C after prolonged equilibration. isolation ofcryoprotein. Centrifugation (50,000 xg, 15 min.) of the cold serum yielded a precipitate with 25 percent. of the E280 nm of whole serum. The precipitate was washed four times with ice-cold phosphate-buffered saline ( 1 3 0 m ~NaCi-lrn~sodium phosphate, p H 7.4) by resuspension and sedimentation as before, and stored in phosphate-buffered saline at 15 mg per ml; the protein reversibly formed a gel in the cold and a clear solution at 37°C. Physico-chemical characterisation of cryoprotein. Immunoelectrophoresis, polyacrylamide gel electrophoresis, sucrose density gradient sedimentation with incorporation of radioactive mouse myeloma protein markers, and reduction and alkylation of cryoglobulin were carried out as described by Parkhouse and Askonas (1969) and Parkhouse, Virella and Dourmashkin (1971). Isoelectric focusing was performed in thin layer polyacrylamide gel (Awdeh, Williamson and Askonas, 1968). Electron miscroscopy. Selected portions of wax-embedded, formalin-fixed blocks of skin and muscle biopsies were taken through xylol and alcohols to 0 1M cacodylate buffer, pH 7.2, post-fixed with 5 per cent. glutaraldehyde and then in 1 per cent. osmium tetroxide, then through alcohols and propylene oxide to Epon embedding. Sections cut at 60-80 nm on an LKB Ultratome 111 were stained by Iead citrate (Venable and Coggeshall, 1965) and uranyl acetate (Hayat, 1969) and examined in the Hitachi HS-8 electron microscope, giving 1 nm resolution. The cryoprecipitated serum at 4°C was dropped from a Pasteur pipette into simultaneous glutaraldehyde-osmium tetroxide fixative at 4°C (Hirsch and Fedorko, 1968), Epon-embedded, sectioned and stained by lead citrate and uranyl acetate. Cryoprecipitate was also fixed in buffered neutral 10 per cent. formalin at 4"C, embedded in paraffin-wax and taken through to Epon and sectioned for electron microscopy as above. Negative-staining of cryoprotein. The procedures used for the preparation of cryoprotein were conventional negative staining methods (Horne, 1971) and the more recent negative staining-carbon technique (Horne and Pasquali-Ronchetti, 1974). Suspensions of isolated cryoprotein prepared according to the methods described above were diluted 1 : 5, 1 : 10, 1 :50 and 1 : 100 with 3 per cent. ammonium molybdatepH 7.0to give a range of concentrations in order to establish optimum spreading conditions in the presence of the negative stain. Mixtures of cryoprotein and negative stain were either spread directly on to carbon filmed grids or mica surface at room temperature (Horne and Pasquali-Ronchetti, 1974). Specimens were examined in a JEOL JEM lOOB electron microscope at 80 kv.
RESULTS The gangrenous leg ulcer The base of the ulcer edge showed chronic and acute inflammatory changes, with a sparse infiltration by lymphocytes and monocytes and concentrations of polymorphonuclear leucocytes; the surface was covered by haemorrhagic
CR YOIMMUNOGLOBULIN IN P YODERMA GANGRENOSUM
27
crust, and inflammatory changes extended to the undermined edge. The lumen of capillaries, venules, and some arterioles in the subcutaneous tissue was distended by homogenous palely-eosinophilic material largely devoid of blood cells; vessel walls and immediately surrounding tissues showed no endothelial swelling or inflammatory cell infiltration. The luminal deposit stained palely with light green, deep purple-blue by PTAH (fig. 1) before and after digestion with fibrinolysin, and red by MSB stain. Congo red staining gave no birefringence for amyloid. The muscle biopsy from the adjacent gastrocnemius showed intact muscle fibres and small blood vessels containing varying amounts of amorphous eosinophilic material, not staining by PTAH or MSB. The bone marrow Four marrow aspirates over 5 mth showed similar, adequate haemopoietic activity. Plasma cell incidence 1-5-34, mean 2.7 per cent. Lymphocytes were below 10 per cent., with 0.5 per cent. of peroxidase-negative monocytoid cells. Peripheral blood and urine The haemoglobin level was 11.6, declining to 8 g per 100 ml, and leucocytes and platelets essentially normal; ESR 52-121 mm per hr (Westergren). The serum acid was 10 mg per 100 ml; blood urea initially 104 mg, declining to 23 mg, but terminally 226 mg per 100 ml. No protein or Bence Jones protein was found in the urine. Kahn, antinuclear factor and human gammaglobulincoated latex fixation tests were negative; cold agglutinins or red cell agglomerating factor were not found, but the Waaler-Rose sheep red cell agglutination test was positive to 32. LE cell tests were negative, and no cryoprotein inclusions found. Serum proteins Serum albumin 2.6 g and plasma fibrinogen 440 mg per 100 ml. Mean total globulins 5.1 g per 100 ml, with the gamma-fraction concentrated in the cryoprecipitate; the range was 4.5-6.5 g, with no tendency to rise over 5 mth, and the separated cryoglobulin was about 2 g per 100 ml. Immunodiffusion indicated that the globulin increase was due to IgG, with IgA and IgM at low normal level; there was no reaction with antifibrinogen serum. Cryoprecipitate and serum viscosity. At 4°C an opaque, bulky precipitate rapidly separated from plasma or serum, with consistency of a gel-like paste. The precipitate deposited on centrifugation at 4°C as a compact mass; further solid phase continued to separate slowly from the clear supernatant at 4°C. At 20°C opaque fluocculent formations slowly separated, proceeding to soft gel formation. Melting to a clear liquid occurred at 29-37°C. Relative viscosities of the serum against water were compared with normal serum after equilibrium at 29", 20" and 30°C (the table); the departure from normal viscosity is parallel to the appearance of cryoprecipitate, and at low temperature this is progressive until a structural gel has formed.
WHITE, A D A M , LAU, HORNE AND PARKHOUSE
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IgGk nature of cryoprecbitate. The purified cryoprotein sedimented in the same position as IgG on sucrose density gradient analysis, and was homogeneous by polyacrylamide gel electrophoresis (fig, 2) and immunoelectrophoresis against rabbit anti-(normal human serum) (fig. 3). Both the sucrose density gradient and polyacrylamide gel analyses were negative for IgM or polymers of IgA. The resolution possible with polyacrylamide gel electrophoresis also indicated an absence of the 7s subunit of IgM (IgMs) in the purified protein. Immunodiffusion against specific antisera identified the protein as IgG with light chain of k type, suggesting a monoclonal origin. The latter was confirmed by isoelectric focusing (fig. 4), which revealed a restricted number of bands, characteristic for individual myeloma proteins (Awdeh et al., 1968) and located in a pH region of 6.6-6.8. TABLE Relative viscosities of serum of normal subject and of the patient in BS/ UIM2 U-tube Ostwald microviscometer after equilibration at various temperatures Viscosity relative to water t"C
Normal serum
Patient's serum
29
1.688
1.645
20
1.736
1.919
4,
1.994 (serum had gelled on long standing; mixed before viscometry) 3
1.826
1.720 (0-5-2 hr) 4 gelling commenced 2.178 (2.5 hr) 4, gel thiikening 3.385 (48 hr)
4,
Complete gelling, reversible at 20°C (72 hr)
Samples of the purified cryoglobulin were reduced at various concentrations of dithioerithritol and subsequently alkylated. The degree of reduction was assessed by electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate, in the presence of which non-covalent interactions between immunoglobulin polypeptide chains and subunits are abolished. Complete reduction into heavy and light chains occurred with dithioerithritol at 5-10 nM and at such concentrations of reducing agent the formation of a cryoprecipitate no longer occurred. At lower concentrations of reducing agent reduction was incomplete, giving a mixture of HzL, H2 and HL, H and L subunits. Cryoprecipitation occurred in such mixtures, but no attempt was made to investigate which subunits retained cryoprecipitability. Opticalproperties of cryoprecipitate. The serum at 4°C showed the presence of birefringent, tactoid-like linear structures. The sections of glutaraldehydeosmium fixed material had a similar appearance in the light microscope when stained by MSB, or particularly by PTAH. In the electron microscope the
CR YOIMMUNOGLOBULIN IN P YODERMA GANGRENOSUM
29
crystallites have a needle-shaped structure (fig. 5 ) comprising parallel tubular units in the long axis (fig. 6), and showing hexagonal packing in cross-section (fig. 7). The wall is apparently composed of electron-dense subunits whereas the tube lumen is non-electron dense; the space between the outer walls of the tubules appears to contain unorganised subunits. The electron micrographs of negatively stained specimens of cryoprotein showed the presence of subunits in the form of various aggregates (fig. 8). In the samples studied there were a number of linear structures present and composed of subunits. These linear structures may have partly formed in solution or assembled during the final dehydration stages of specimen preparation, but their appearance and dimensions were similar to the individual linear components observed in thin sections showing tubular assemblies (figs. 5, 6, 7). In addition to the linear structures, many areas contained random aggregates of structural components or subunits interpreted as being the cryoprotein macromolecules (fig. 8). The possibility of assembling cryoprotein in vitro to form linear arrays or microtubular components requires further investigation and is obviously a subject beyond the scope of this communication. Electron microscopy of skin biopsy of ulcer Vascular lumen. Sections from throughout the biopsy showed that the vessels were distended by electron-dense material with a high degree of ordered structure similar to the tubes in the serum cryoprotein crystallites (figs. 9-1 1). The protein extended to the endothelial lining of the vessels with variable orientation; there was no endothelial cell or other vessel-wall damage, or cellular infiltration (fig. 9). Extravascular tissues. Similar oriented protein with tubular structure was found outside the blood vessels, lying free as well as to a limited extent within the cytoplasm of macrophages, and particularly abundant at the ulcer base (fig. 12). Muscle biopsy. Small vessels in the biopsy from adjacent gastrocnemius contained some amorphous material, but there was no trace of the oriented tubular structure. Dimensions of the tubular assemblies Calibrated photographs of the tubes were measured in longitudinal and cross-section in sections of the cryoprecipitated serum and of the intra- and extravascular areas of the skin biopsy. The mean external diameter of the tubes is 18.2 nm (SD 2.4 nm; n = 210). The mean internal diameter is 8-5 nm (SD 1-9 nm; n = 210). The wall thickness by difference is 4.85 nm, with SD 0.89 nm by error analysis. The distance apart of the hexagonally-packed tubes is 8.2 nm (SD 1.5 nm; n = 50). The maximum length of the tubes is not known, but individual crystallites with length up to about 5 p m are apparent by light microscopy.
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WHITE, ADAM, LAU, HORNE AND PARKHOUSE
DISCUSSION Pyoderma gangrenosum Pyoderma gangrenosum can be associated with a variety of underlying causes, particularly imunopathological disorders (Thompson et al., 1973). Various dermatological abnormalities occur with circulating cryoproteins, including gangrenous ulcers (Feldaker et al., 1956; Baughman and Sommer, 1966). In the present case the vascular obstruction was due to deposition of the cryoglobulin alone as tubular micro-crystallites, and the vessel wall appeared intact. Similar deposits were found outside the vessels, and it appears likely that these were built up from the IgGk monomer in situ. The MSB and PTAH stains gave positive reactions within, and in some areas without the vessels of the ulcer. The presence of fibrin in intravascular deposits would seem to be a possibility, but no fibrin was identified by electron microscopy, and the tubular crystallites from the fibrinogen/fibrin-free serum gave similar staining. However, no immunofluorescent investigations were made for the possible presence of small amounts of fibrin, complement components, or immunoglobulins other than the IgGk tubular assembles. Bogaars et al. (1973) found no immunological evidence for the precipitation of other serum proteins in the building of tubular units in vitro from a similar myeloma IgGk cryoglobulin. Scanty deposits within the vessels in the adjacent muscle biopsy showed no tubular assemblies. Ellis (1964) and Baughman and Sommer (1966) described patients with cryomacroglobulinaemias, and showed ulcerated lesions with distension of small vessels by homogeneous eosinophilic material by light microscopy and apparently intact vascular walls, but without ultrastructural information; in the second authors’ case there were also extravascular eosinophilic deposits. Monoclonal cryoglobulin in relation to myeloma Cryoimmunoglobulin is associated with some myelomas, and monoclonal cryoglobulinaemias without malignant features may terminate after years in frank myeloma (Zawadzki and Edwards, 1972). Gordon-Smith et al. (1968) described a patient with ulceration of hands and feet and 3-5 per cent. of marrow plasma cells associated with crystalline IgGl cryoglobulin; 14 years later there was overt myeloma. During the short period of observation of the present case overt evidence of myeloma was not obtained, though plasma cells are likely to have been the source of the cryoglobulin. Cryoglobulins and disturbed blood rheology Hyperviscosity syndromes may be associated with various immunoglobulin abnormalities and Buxbaum (1972) has considered ways in which abnormal IgG monoclonal globulins affect viscosity. Anomalous increase with rise in
CR YOIMMUNOGLOBULIN IN P YODERMA GANGRENOSUM
31
protein concentration is more marked with IgM than IgG (Fahey et al., 1965), but molecular shape is important in IgG (MacKenzie et al., 1970) as the IgG3 subclass particularly shows marked aggregation to large polymers with increased viscosity (Capra and Kunkel, 1970). In cryoglobulinaemias, quite abrupt changes in rheological behaviour may be expected with cooling. Saha et al. (1968) related IgGI cryoglobulin structure to cryoprecipitation, dependent upon pH and ionic and non-ionic environment as well as temperature; the abnormal globulin had relatively more hydrophobic non-polar amino acid groups than normal IgG, and cryopreciptation appeared to reflect inadequqacy in water : protein interaction. Temperature effects on the serum in the present case were studied at a single low rate of shear, and non-Newtonian behaviour is not known. However, it is apparent from the table that before precipitation the serum viscosity does not differ from normal, that separation begins slowly with moderate cooling, and that at low temperature it proceeds to complete, reversible gelation. The birefringent, tactoid-like domains ultimately build extensive arrays of ordered, regular tubules. Tubule formation may be accompanied by building of spherical and other assemblies from IgGk monomer, as visualised in the serum by negative-staining. The relatively static circulation in the skin at the ulcer site may favour stability of the completed arrays and lead to irreversible obstruction. Tubular protein structures Certain proteins such as beef catalase (Kiselev et al., 1967), cytochrome P-420, normal and the oxy- form of sickle haemoglobin (White and Heagan, 1970), and some larger enzymes can assemble in microtubular structures (Caspar and Klug, 1962), and Home (1971) has reviewed the use of negative staining as complementary to thin-section electron microscopy and diffraction for their elucidation. Both plant viruses (Bancroft et al., 1967; Hull et al., 1970) and some human and animal viruses may occur in aberrant or elongated forms, based on pentameric or hexameric helical protein assemblies. Such assemblies may occur with variants of normally monomeric proteins, and Bogaars et al. (1973) have described a human myeloma IgGk cryoglobulin with tubular crystalline structure which appears to be essentially similar to the cryoglobulin we describe. The wall thickness is very similar for the two proteins, though the measurement of external and internal diameter of the tubes are smaller in our case. They consider that the tubular units could accommodate 10 or 12 Fab fragments, representing five or six T or shallow Y-shaped molecules bonded together in circular arrangement in cross-sectional view, though with some flexion at the hinge region between Fab fragment pairs. The assumption of stable rod-like forms is related to the shape of the unit molecules and interaction forces between them, with a tube-like inner cavity where the molecules are smaller than the radius of the cylinder (Kiselev et al., 1967). These conditions appear to be present in the case of IgGk cryoglobulins described by Bogaars et al. (1973) and by ourselves, and may lead directly to pathological consequences.
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WHITE, ADAM, LAU, HORNE AND PARKHOUSE
sW
R
Y
Recurrent gangrenous leg ulcers were associated with a monoclonal IgGIc cryoglobulinaemia, though without overt myeloma. Cryoprecipitation of the serum in vitro gave a reversible gel with microtubular structure, built from the monomer and with a high degree of order. The same structure was found distending the superficial vessels of the ulcer biopsy, as well as extravascularly. There was no vasculitis, and luminal obstruction appeared responsible for the ulceration. Microtubular structures occur in nature among diverse proteins and viruses. The protein studied here is similar to a reported instance of myeloma cryo-IgGk. We wish to thank Assoc. Prof. H. 0. Wong, Head, Dept. of Medicine, University of Malaya, for permission to study this case; Electron Microscopist, Mr K. L. Chong: Mr N. A. Nathan and K. H. Yee for valuable assistance; and Miss Jenny Chay for secretarial assistance; and Profs. R. Markham and R. D. Preston for their interest. One of us (J. C. W.) is in receipt of a Special Commonwealth Award. REFERENCES A. R., AND ASKONAS, B. A. 1968. Isoelectric focussing in polyAWDEn, Z . L., WILLIAMSON, acrylamide gel and its application to immunoglobulins. Nature, Lond., 219, 66. BANCROFT, J. N., HILLS,G. J., AND MARKHAM, R. 1967. A study of the self-assembly process in a small spherical virus. Formation of organized structures from protein subunits in vitro. Virology,31, 354. R. D., AND SOMMER, R. G. 1966. Cryoglobulinemia presenting as " factitial BAUGHMAN, ulceration ". Arch. Drem., 94, 725. H. A., KALDERON, A. E., CUMMINGS, F. J., KAPLAN,S., MELNICOF, I., PARK,C., BOGAARS, DIAMOND, I., AND CALABRESI, P. 1973. Human IgG cryoglobulin with tubular crystal structure. Nature, New Biol., 245, 117. BUXBAIJM,J. 1972. Perspective: hyperviscosity syndrome in dysproteinemias. Amer. J. med. Sci., 264, 123 H. J. 1970. Aggegation of yG3 proteins: relevance to the hyperCAPRA,J. D., AND KUNKEL, viscosity syndrome. J. clin. Invest., 49, 610. CARTER,P. M. 1973. Occasional survey: immune complex diseases. Ann. rheum. Dis., 32, 265. CASPAR,C. L. D., AND KLUG,A. 1962. Physical principles in the construction of regular viruses. Cold Spring Harbor Symp. Quanti. Biol., 21, 1. ELLIS,F. A. 1964. The cutaneous manifestations of cryoglobulinemia. Archs. Derm., 89, 690. FAHEY, J. L., BARTH,W. F., AND SOLOMON, A. 1965. Serum hyperviscosity syndrome. J. Amer. Med. Assoc., 192, 120. FELDAKER, M., PERRY, H. O., AND HANLON,D. G . 1956. Dermatological manifestations associated with cryoglobulinemia. Arch. Der., 73, 325. E. C., HARRISON,R. J., AND HOBBS,J. R. 1968. Hyperproteinaemia GORDON-SMITH, associated with multiple myeloma. Proc. roy. SOC.Med., 61, 1112. P. F. 1973. Cryoimmunoglobulins. Sem. Hematol., 10, 87. GREY,H. M., AND KOHLER, HAYAT,M. A. 1969. Uranyl acetate as a stain and fixative for heart tissue. Proc. 27th Ann. Meet. Electron Micros. SOC.Am., Baton Rouge, p. 412. HIRSCH,J. G., AND FEDORKO, M. E. 1968. Ultrastructure of human leukocytes after simultaneous fixation in glutaraldehyde and osmium tetroxide and " post fixation " in uranyl acetate. J. Cell. Biol., 38, 615. HORNE,R. W. 1971. Electron microscopy applied to the study of macromolecular components assembled to form complex biological structures. symp. SOC.Exp. Biol., 25,71.
WHITE,ADAM,LAU,HORNE AND PARKHOUSE CRYOIMMUNOGLOBULIN IN PYODERMA
FIG.1.-Arterioles
PLATEVI GANGRENOSUM
in the dermis distended by protein, adjacent to area of purulent infiltration. Phosphotungstic acid haematoxylin (PTAH). x 480.
albumin IgG IgMs IgM FIG.2.-Analysis of acrylamide gel electrophoresis. ( a ) whole serum containing the cryoglobulin, (b) the isolated cryoglobulin. The position of marker proteins run in parallel is indicated on the figure.
FIG.3.-Tmmunoelectrophoretic analysis. ( a ) whole serum containing the cryoglobulin, (b) the isolated cryoglobulin. The upper two troughs contain anti-(normal human serum), and the lower one anti-human Fc isolated from IgG. Cathode on the left.
WHITE,ADAM,LAU, HORNEAND PARKHOUSE
VII PLATE
CRYOIMMUNOGLOBULIN I N PYODERMAGANGRENOSUM
FIG.4.-Isoelectric focusing of the isolated cryoglobulin. (a) (above) the isolated cryoglobulin, ( h ) (below)a monoclonal mouse IgG2. isolated from the serum of mice bearing the plasma cell tuiiiour Adj P C 5 .
FIG.5.--Crystallites of cryoglobulin in serum fixed at 4°C in simultaneous glutaraldehyde-osmium tetroxide, showing mainly longitudinal section of tubules. x 53,000. FIGS.5-7 are from electron micrographs stained by lead citrate and uranyl acetate.
W H I T E , ADAM,LAU, HORNE AND
PARKHOUSE
PLATEVIII
CRYOIMMUNOGLOBULIN IN PYODERMA GANGRENOSUM
FIG.6.-Parallel tubular units seen in longitudinal section in cryoprecipitated serum.
FIG. 7.-Cross-section
x 160,000.
of hexagonally-packed tubular units in cryoprecipitated serum, showing indication of structural subunits in and between the walls, and non-electron-dense lumina. x 160,000.
WIIITE, A D A M ,
LAU,HORNEAND
PARKHOUSE
PLATEIX
CRYOIMMUNOGLOBULIN IN PYODERMA GANGRENOSUM
FIG.8.-Electron micrograph of negatively stained cryoprotein. The small particles (arrows '' A ") correspond to the subunits observed as isolated components or in random aggre$?tes. Many areas of the specimens examined contained linear arrays of the type indicated at " B . x 60,oqO. Insert: high-magnification region showing groups of subunits associated with cryoprotern. x 230,000.
WHITE,ADAM,LAU, HORNEAND PARKHOUSE
PLATE X
CRYOIMMUNOGLOBULIN I N PYODERMA GANGRENOSUM
FIG.9.-Cross-section of small vessel in the dermis of skin biopsy, showing the lumen distended by precipitated protein, in which organised structure is visible. x 16,800.
FIG.
higher magnification of vessel contents, showing masses of hexagonally-packed tubules in cross-section. x 53,000. FIGS.9-12 are from electron micrographs stained by lead citrate and uranyl acetate.
101-A
WHITE,
ADAM,LAU, HORNE AND PARKHOUSE
PLATEXI
CRYOIMMUNOGLOBULIN I N PYODERMA GANGRENOSUM
FIG.
FIG.
I I .-Section
exhibiting various orientations of intravascular protein tubular assemblies. x 53,000.
12.-Protein masses with tubular structure lying in extravascular tissue in the dermis.
X
53,000.
33
CR YOIMMUNOGLOBULIN IN PYODERMA GANGRENOSUM
HORNE,R. W., AND PASQUALI-RONCHETTI, I. P. 1974. A negative staining-carbon film technique for studying viruses in the electron microscope. I. Preparative procedures for examining icosahedral and filamentous viruses. J. Ultrastruct. Res., 41, 361. A. 1970. The in vivo behaviour of twenty-four strains HULL,R., HILLS,G. J., AND FLASKITT, of Alfalfa mosaic virus. Virology, 42, 753. c. L., AND VAINSHTEIN,B. K. 1967. Crystallization of catalase KISELEV, N. A,, SHPITZBERG, in the form of tubes with mono-molecular walls. J. Mol. Biol., 25, 433. H. H., AND OREILLY,R. A. 1970. The hyperviscosity MACKENZIE,M. R. FUDENBERG, syndrome. I. In IgG myeloma. The role of protein concentration and molecular shape. J . Clin. Invest., 49, 15. MOROZ,L. A., AND ROSE,B. 1971. In Immunological diseases, 2nd ed., edited by M. Samter, Boston, vol. 1, p. 459. PARKHOUSE, R. M. E., AND ASKONAS, B. A. 1969. Immunoglobulin M biosynthesis. Intracellular accumulation of 7 s sbunits. Biochem. J., 115, 163. PARKHOUSE, R. M. E., VIRELLA, G., AND DOURMASHKIN, R. R. 1971. Structural characterization of a human monoclonal IgA protein. Clin. exp. Immunol., 8, 581. SAHA,A., EDWARDS, M. A., SARGENT, A. U., AND ROSE,B. 1968. Mechanism of cryoprecipitation. I. Characteristics of a human cryoglobulin. Zmmunochemistry, 5 , 341. D. M., MAIN,R. A., BECK,J. S., AND ALBERT-RECHT, F. 1973. Studies on a THOMPSON, patient with leucocytoclastic vasculitis, ‘‘ pyoderma gangrenosum ” and paraproteinaemia. Br. J. Derrn., 88, 117. H. H., AND COGGESHALL, R. 1965. A simplified lead citrate stain for use in electron VENABLE, microscopy. J. Cell. Biol., 25, 407. WELLS,R. 1970. Medical intelligence: syndromes of hyperviscosity. New Engl. J. Med., 283, 183. WHITE,J. G., AND HEAGAN, B. 1970. Tubular polymers of normal human hemoglobin. Amer. J. Path., 59, 101. Z. A., AND EDWARDS,G. A. 1972. Nonmyelomatous monoclonal immunoZAWADZKI, globulinemia. Progr. Clin. Immunol., 1, 105.
J. PATH.-VOL.
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R. W. HORNE Jolm Innes Institute, Norwich, England
R. M. E. PARKHOUSE National Institute for Medical Research, Mill Hill, London NW7, England
PLATES VI-XI U L C E R A T I OofNthe leg with Raynaud’s phenomenon can mark the presence of paraproteinaemia, including cryoglobulinaemia (Ellis, 1964), with altered blood viscosity and flow (Wells, 1970). Cryoglobulins may form intravascular deposits in the cold with local ischaemia, or complexes may be formed within the vessel wall with associated vasculitis; the cryoproteins may be monoclonal or multicomponent (Moroz and Rose, 1971), and the mixed forms may be immune-complexes (Carter, 1973). Monoclonal cryoglobulins are particularly associated with plasma cell and lympho-reticular proliferative conditions, and crystalline structure has been observed in some (Grey and Kohler, 1973). A particular tubular crystalline structure has been reported in a case of IgGk myeloma (Bogaars et al., 1973). Tubular protein structures appear to be of quite widespread and fundamental natural occurrence (Horne, 1971), and we report studies on a IgGk cryoglobulin with this characteristic, occurring in a patient with pyoderma gangrenosum of the leg but without overt myeloma; vascular luminal obstruction appears to underlie the ulceration. MATERIALS AND METHODS Case report A Chinese man of 64 had painful recurrent ulcers on both calves for 3 y, healing with hyperpigmented scars, but progressing to a large (10 x 3 cm) gangrenous ulcer on the right. On cold exposure both legs became blue and painful, with ankle petechiae. All peripheral pulses were felt and blood pressure was 150/90 mm Hg. Pyoderma gangrenosum was diagnosed. Gelling of the serum occurred at 4°C. The bone marrow was not indicative of meyloma. A benign ulcer was present at the pyloric antrum. Over 6 mth his leg ulcers healed with oral prednisolone and later dapsone, though cold intolerance persisted. At this time collapse of L1 and L5 vertebrae was found, though the rest of the skeleton was normal and three further marrow aspirations showed low plasma Received 17 Sept. 1975; accepted 7 Oct. 1975. 1. PATH.-VOL.
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WHITE, ADAM, LAU, HORNE AND PARKHOUSE
cell incidence. Two months later he was admitted in terminal coma with reappearance of calf ulceration and gangrene of fingers. Necropsy was not obtained. Investigations Histological and cytological methods. Biopsies from ulcer edge and gastrocnemius muscle beneath intact skin were formalin-fixed, paraffin-embedded, and sections stained by haematoxylin and eosin, light green and nuclear fast red, Congo red, Mallory's phosphotungstic acid haematoxylin (PTAH ; before and after fibrinolysin treatment) and Martius-ScarletBlue (MSB) stain. Aspiration and needle-trephine biopsies of iliac crest and sternal bone marrow were stained by May-Grunwald-Giemsa, Prussian-blue and PAS. Serum protein investigation. Cellulose acetate electrophoresis was performed in barbitone buffer, pH 8.6; the cryoprecipitate and supernatant separated by centrifugation at 4°C were run separately after melting at room temperature. Relative viscosity of whole serum was determined in a BS/U/M2 U-tube Ostwald microviscometer a t 29", 20" and 3°C after prolonged equilibration. isolation ofcryoprotein. Centrifugation (50,000 xg, 15 min.) of the cold serum yielded a precipitate with 25 percent. of the E280 nm of whole serum. The precipitate was washed four times with ice-cold phosphate-buffered saline ( 1 3 0 m ~NaCi-lrn~sodium phosphate, p H 7.4) by resuspension and sedimentation as before, and stored in phosphate-buffered saline at 15 mg per ml; the protein reversibly formed a gel in the cold and a clear solution at 37°C. Physico-chemical characterisation of cryoprotein. Immunoelectrophoresis, polyacrylamide gel electrophoresis, sucrose density gradient sedimentation with incorporation of radioactive mouse myeloma protein markers, and reduction and alkylation of cryoglobulin were carried out as described by Parkhouse and Askonas (1969) and Parkhouse, Virella and Dourmashkin (1971). Isoelectric focusing was performed in thin layer polyacrylamide gel (Awdeh, Williamson and Askonas, 1968). Electron miscroscopy. Selected portions of wax-embedded, formalin-fixed blocks of skin and muscle biopsies were taken through xylol and alcohols to 0 1M cacodylate buffer, pH 7.2, post-fixed with 5 per cent. glutaraldehyde and then in 1 per cent. osmium tetroxide, then through alcohols and propylene oxide to Epon embedding. Sections cut at 60-80 nm on an LKB Ultratome 111 were stained by Iead citrate (Venable and Coggeshall, 1965) and uranyl acetate (Hayat, 1969) and examined in the Hitachi HS-8 electron microscope, giving 1 nm resolution. The cryoprecipitated serum at 4°C was dropped from a Pasteur pipette into simultaneous glutaraldehyde-osmium tetroxide fixative at 4°C (Hirsch and Fedorko, 1968), Epon-embedded, sectioned and stained by lead citrate and uranyl acetate. Cryoprecipitate was also fixed in buffered neutral 10 per cent. formalin at 4"C, embedded in paraffin-wax and taken through to Epon and sectioned for electron microscopy as above. Negative-staining of cryoprotein. The procedures used for the preparation of cryoprotein were conventional negative staining methods (Horne, 1971) and the more recent negative staining-carbon technique (Horne and Pasquali-Ronchetti, 1974). Suspensions of isolated cryoprotein prepared according to the methods described above were diluted 1 : 5, 1 : 10, 1 :50 and 1 : 100 with 3 per cent. ammonium molybdatepH 7.0to give a range of concentrations in order to establish optimum spreading conditions in the presence of the negative stain. Mixtures of cryoprotein and negative stain were either spread directly on to carbon filmed grids or mica surface at room temperature (Horne and Pasquali-Ronchetti, 1974). Specimens were examined in a JEOL JEM lOOB electron microscope at 80 kv.
RESULTS The gangrenous leg ulcer The base of the ulcer edge showed chronic and acute inflammatory changes, with a sparse infiltration by lymphocytes and monocytes and concentrations of polymorphonuclear leucocytes; the surface was covered by haemorrhagic
CR YOIMMUNOGLOBULIN IN P YODERMA GANGRENOSUM
27
crust, and inflammatory changes extended to the undermined edge. The lumen of capillaries, venules, and some arterioles in the subcutaneous tissue was distended by homogenous palely-eosinophilic material largely devoid of blood cells; vessel walls and immediately surrounding tissues showed no endothelial swelling or inflammatory cell infiltration. The luminal deposit stained palely with light green, deep purple-blue by PTAH (fig. 1) before and after digestion with fibrinolysin, and red by MSB stain. Congo red staining gave no birefringence for amyloid. The muscle biopsy from the adjacent gastrocnemius showed intact muscle fibres and small blood vessels containing varying amounts of amorphous eosinophilic material, not staining by PTAH or MSB. The bone marrow Four marrow aspirates over 5 mth showed similar, adequate haemopoietic activity. Plasma cell incidence 1-5-34, mean 2.7 per cent. Lymphocytes were below 10 per cent., with 0.5 per cent. of peroxidase-negative monocytoid cells. Peripheral blood and urine The haemoglobin level was 11.6, declining to 8 g per 100 ml, and leucocytes and platelets essentially normal; ESR 52-121 mm per hr (Westergren). The serum acid was 10 mg per 100 ml; blood urea initially 104 mg, declining to 23 mg, but terminally 226 mg per 100 ml. No protein or Bence Jones protein was found in the urine. Kahn, antinuclear factor and human gammaglobulincoated latex fixation tests were negative; cold agglutinins or red cell agglomerating factor were not found, but the Waaler-Rose sheep red cell agglutination test was positive to 32. LE cell tests were negative, and no cryoprotein inclusions found. Serum proteins Serum albumin 2.6 g and plasma fibrinogen 440 mg per 100 ml. Mean total globulins 5.1 g per 100 ml, with the gamma-fraction concentrated in the cryoprecipitate; the range was 4.5-6.5 g, with no tendency to rise over 5 mth, and the separated cryoglobulin was about 2 g per 100 ml. Immunodiffusion indicated that the globulin increase was due to IgG, with IgA and IgM at low normal level; there was no reaction with antifibrinogen serum. Cryoprecipitate and serum viscosity. At 4°C an opaque, bulky precipitate rapidly separated from plasma or serum, with consistency of a gel-like paste. The precipitate deposited on centrifugation at 4°C as a compact mass; further solid phase continued to separate slowly from the clear supernatant at 4°C. At 20°C opaque fluocculent formations slowly separated, proceeding to soft gel formation. Melting to a clear liquid occurred at 29-37°C. Relative viscosities of the serum against water were compared with normal serum after equilibrium at 29", 20" and 30°C (the table); the departure from normal viscosity is parallel to the appearance of cryoprecipitate, and at low temperature this is progressive until a structural gel has formed.
WHITE, A D A M , LAU, HORNE AND PARKHOUSE
28
IgGk nature of cryoprecbitate. The purified cryoprotein sedimented in the same position as IgG on sucrose density gradient analysis, and was homogeneous by polyacrylamide gel electrophoresis (fig, 2) and immunoelectrophoresis against rabbit anti-(normal human serum) (fig. 3). Both the sucrose density gradient and polyacrylamide gel analyses were negative for IgM or polymers of IgA. The resolution possible with polyacrylamide gel electrophoresis also indicated an absence of the 7s subunit of IgM (IgMs) in the purified protein. Immunodiffusion against specific antisera identified the protein as IgG with light chain of k type, suggesting a monoclonal origin. The latter was confirmed by isoelectric focusing (fig. 4), which revealed a restricted number of bands, characteristic for individual myeloma proteins (Awdeh et al., 1968) and located in a pH region of 6.6-6.8. TABLE Relative viscosities of serum of normal subject and of the patient in BS/ UIM2 U-tube Ostwald microviscometer after equilibration at various temperatures Viscosity relative to water t"C
Normal serum
Patient's serum
29
1.688
1.645
20
1.736
1.919
4,
1.994 (serum had gelled on long standing; mixed before viscometry) 3
1.826
1.720 (0-5-2 hr) 4 gelling commenced 2.178 (2.5 hr) 4, gel thiikening 3.385 (48 hr)
4,
Complete gelling, reversible at 20°C (72 hr)
Samples of the purified cryoglobulin were reduced at various concentrations of dithioerithritol and subsequently alkylated. The degree of reduction was assessed by electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate, in the presence of which non-covalent interactions between immunoglobulin polypeptide chains and subunits are abolished. Complete reduction into heavy and light chains occurred with dithioerithritol at 5-10 nM and at such concentrations of reducing agent the formation of a cryoprecipitate no longer occurred. At lower concentrations of reducing agent reduction was incomplete, giving a mixture of HzL, H2 and HL, H and L subunits. Cryoprecipitation occurred in such mixtures, but no attempt was made to investigate which subunits retained cryoprecipitability. Opticalproperties of cryoprecipitate. The serum at 4°C showed the presence of birefringent, tactoid-like linear structures. The sections of glutaraldehydeosmium fixed material had a similar appearance in the light microscope when stained by MSB, or particularly by PTAH. In the electron microscope the
CR YOIMMUNOGLOBULIN IN P YODERMA GANGRENOSUM
29
crystallites have a needle-shaped structure (fig. 5 ) comprising parallel tubular units in the long axis (fig. 6), and showing hexagonal packing in cross-section (fig. 7). The wall is apparently composed of electron-dense subunits whereas the tube lumen is non-electron dense; the space between the outer walls of the tubules appears to contain unorganised subunits. The electron micrographs of negatively stained specimens of cryoprotein showed the presence of subunits in the form of various aggregates (fig. 8). In the samples studied there were a number of linear structures present and composed of subunits. These linear structures may have partly formed in solution or assembled during the final dehydration stages of specimen preparation, but their appearance and dimensions were similar to the individual linear components observed in thin sections showing tubular assemblies (figs. 5, 6, 7). In addition to the linear structures, many areas contained random aggregates of structural components or subunits interpreted as being the cryoprotein macromolecules (fig. 8). The possibility of assembling cryoprotein in vitro to form linear arrays or microtubular components requires further investigation and is obviously a subject beyond the scope of this communication. Electron microscopy of skin biopsy of ulcer Vascular lumen. Sections from throughout the biopsy showed that the vessels were distended by electron-dense material with a high degree of ordered structure similar to the tubes in the serum cryoprotein crystallites (figs. 9-1 1). The protein extended to the endothelial lining of the vessels with variable orientation; there was no endothelial cell or other vessel-wall damage, or cellular infiltration (fig. 9). Extravascular tissues. Similar oriented protein with tubular structure was found outside the blood vessels, lying free as well as to a limited extent within the cytoplasm of macrophages, and particularly abundant at the ulcer base (fig. 12). Muscle biopsy. Small vessels in the biopsy from adjacent gastrocnemius contained some amorphous material, but there was no trace of the oriented tubular structure. Dimensions of the tubular assemblies Calibrated photographs of the tubes were measured in longitudinal and cross-section in sections of the cryoprecipitated serum and of the intra- and extravascular areas of the skin biopsy. The mean external diameter of the tubes is 18.2 nm (SD 2.4 nm; n = 210). The mean internal diameter is 8-5 nm (SD 1-9 nm; n = 210). The wall thickness by difference is 4.85 nm, with SD 0.89 nm by error analysis. The distance apart of the hexagonally-packed tubes is 8.2 nm (SD 1.5 nm; n = 50). The maximum length of the tubes is not known, but individual crystallites with length up to about 5 p m are apparent by light microscopy.
30
WHITE, ADAM, LAU, HORNE AND PARKHOUSE
DISCUSSION Pyoderma gangrenosum Pyoderma gangrenosum can be associated with a variety of underlying causes, particularly imunopathological disorders (Thompson et al., 1973). Various dermatological abnormalities occur with circulating cryoproteins, including gangrenous ulcers (Feldaker et al., 1956; Baughman and Sommer, 1966). In the present case the vascular obstruction was due to deposition of the cryoglobulin alone as tubular micro-crystallites, and the vessel wall appeared intact. Similar deposits were found outside the vessels, and it appears likely that these were built up from the IgGk monomer in situ. The MSB and PTAH stains gave positive reactions within, and in some areas without the vessels of the ulcer. The presence of fibrin in intravascular deposits would seem to be a possibility, but no fibrin was identified by electron microscopy, and the tubular crystallites from the fibrinogen/fibrin-free serum gave similar staining. However, no immunofluorescent investigations were made for the possible presence of small amounts of fibrin, complement components, or immunoglobulins other than the IgGk tubular assembles. Bogaars et al. (1973) found no immunological evidence for the precipitation of other serum proteins in the building of tubular units in vitro from a similar myeloma IgGk cryoglobulin. Scanty deposits within the vessels in the adjacent muscle biopsy showed no tubular assemblies. Ellis (1964) and Baughman and Sommer (1966) described patients with cryomacroglobulinaemias, and showed ulcerated lesions with distension of small vessels by homogeneous eosinophilic material by light microscopy and apparently intact vascular walls, but without ultrastructural information; in the second authors’ case there were also extravascular eosinophilic deposits. Monoclonal cryoglobulin in relation to myeloma Cryoimmunoglobulin is associated with some myelomas, and monoclonal cryoglobulinaemias without malignant features may terminate after years in frank myeloma (Zawadzki and Edwards, 1972). Gordon-Smith et al. (1968) described a patient with ulceration of hands and feet and 3-5 per cent. of marrow plasma cells associated with crystalline IgGl cryoglobulin; 14 years later there was overt myeloma. During the short period of observation of the present case overt evidence of myeloma was not obtained, though plasma cells are likely to have been the source of the cryoglobulin. Cryoglobulins and disturbed blood rheology Hyperviscosity syndromes may be associated with various immunoglobulin abnormalities and Buxbaum (1972) has considered ways in which abnormal IgG monoclonal globulins affect viscosity. Anomalous increase with rise in
CR YOIMMUNOGLOBULIN IN P YODERMA GANGRENOSUM
31
protein concentration is more marked with IgM than IgG (Fahey et al., 1965), but molecular shape is important in IgG (MacKenzie et al., 1970) as the IgG3 subclass particularly shows marked aggregation to large polymers with increased viscosity (Capra and Kunkel, 1970). In cryoglobulinaemias, quite abrupt changes in rheological behaviour may be expected with cooling. Saha et al. (1968) related IgGI cryoglobulin structure to cryoprecipitation, dependent upon pH and ionic and non-ionic environment as well as temperature; the abnormal globulin had relatively more hydrophobic non-polar amino acid groups than normal IgG, and cryopreciptation appeared to reflect inadequqacy in water : protein interaction. Temperature effects on the serum in the present case were studied at a single low rate of shear, and non-Newtonian behaviour is not known. However, it is apparent from the table that before precipitation the serum viscosity does not differ from normal, that separation begins slowly with moderate cooling, and that at low temperature it proceeds to complete, reversible gelation. The birefringent, tactoid-like domains ultimately build extensive arrays of ordered, regular tubules. Tubule formation may be accompanied by building of spherical and other assemblies from IgGk monomer, as visualised in the serum by negative-staining. The relatively static circulation in the skin at the ulcer site may favour stability of the completed arrays and lead to irreversible obstruction. Tubular protein structures Certain proteins such as beef catalase (Kiselev et al., 1967), cytochrome P-420, normal and the oxy- form of sickle haemoglobin (White and Heagan, 1970), and some larger enzymes can assemble in microtubular structures (Caspar and Klug, 1962), and Home (1971) has reviewed the use of negative staining as complementary to thin-section electron microscopy and diffraction for their elucidation. Both plant viruses (Bancroft et al., 1967; Hull et al., 1970) and some human and animal viruses may occur in aberrant or elongated forms, based on pentameric or hexameric helical protein assemblies. Such assemblies may occur with variants of normally monomeric proteins, and Bogaars et al. (1973) have described a human myeloma IgGk cryoglobulin with tubular crystalline structure which appears to be essentially similar to the cryoglobulin we describe. The wall thickness is very similar for the two proteins, though the measurement of external and internal diameter of the tubes are smaller in our case. They consider that the tubular units could accommodate 10 or 12 Fab fragments, representing five or six T or shallow Y-shaped molecules bonded together in circular arrangement in cross-sectional view, though with some flexion at the hinge region between Fab fragment pairs. The assumption of stable rod-like forms is related to the shape of the unit molecules and interaction forces between them, with a tube-like inner cavity where the molecules are smaller than the radius of the cylinder (Kiselev et al., 1967). These conditions appear to be present in the case of IgGk cryoglobulins described by Bogaars et al. (1973) and by ourselves, and may lead directly to pathological consequences.
32
WHITE, ADAM, LAU, HORNE AND PARKHOUSE
sW
R
Y
Recurrent gangrenous leg ulcers were associated with a monoclonal IgGIc cryoglobulinaemia, though without overt myeloma. Cryoprecipitation of the serum in vitro gave a reversible gel with microtubular structure, built from the monomer and with a high degree of order. The same structure was found distending the superficial vessels of the ulcer biopsy, as well as extravascularly. There was no vasculitis, and luminal obstruction appeared responsible for the ulceration. Microtubular structures occur in nature among diverse proteins and viruses. The protein studied here is similar to a reported instance of myeloma cryo-IgGk. We wish to thank Assoc. Prof. H. 0. Wong, Head, Dept. of Medicine, University of Malaya, for permission to study this case; Electron Microscopist, Mr K. L. Chong: Mr N. A. Nathan and K. H. Yee for valuable assistance; and Miss Jenny Chay for secretarial assistance; and Profs. R. Markham and R. D. Preston for their interest. One of us (J. C. W.) is in receipt of a Special Commonwealth Award. REFERENCES A. R., AND ASKONAS, B. A. 1968. Isoelectric focussing in polyAWDEn, Z . L., WILLIAMSON, acrylamide gel and its application to immunoglobulins. Nature, Lond., 219, 66. BANCROFT, J. N., HILLS,G. J., AND MARKHAM, R. 1967. A study of the self-assembly process in a small spherical virus. Formation of organized structures from protein subunits in vitro. Virology,31, 354. R. D., AND SOMMER, R. G. 1966. Cryoglobulinemia presenting as " factitial BAUGHMAN, ulceration ". Arch. Drem., 94, 725. H. A., KALDERON, A. E., CUMMINGS, F. J., KAPLAN,S., MELNICOF, I., PARK,C., BOGAARS, DIAMOND, I., AND CALABRESI, P. 1973. Human IgG cryoglobulin with tubular crystal structure. Nature, New Biol., 245, 117. BUXBAIJM,J. 1972. Perspective: hyperviscosity syndrome in dysproteinemias. Amer. J. med. Sci., 264, 123 H. J. 1970. Aggegation of yG3 proteins: relevance to the hyperCAPRA,J. D., AND KUNKEL, viscosity syndrome. J. clin. Invest., 49, 610. CARTER,P. M. 1973. Occasional survey: immune complex diseases. Ann. rheum. Dis., 32, 265. CASPAR,C. L. D., AND KLUG,A. 1962. Physical principles in the construction of regular viruses. Cold Spring Harbor Symp. Quanti. Biol., 21, 1. ELLIS,F. A. 1964. The cutaneous manifestations of cryoglobulinemia. Archs. Derm., 89, 690. FAHEY, J. L., BARTH,W. F., AND SOLOMON, A. 1965. Serum hyperviscosity syndrome. J. Amer. Med. Assoc., 192, 120. FELDAKER, M., PERRY, H. O., AND HANLON,D. G . 1956. Dermatological manifestations associated with cryoglobulinemia. Arch. Der., 73, 325. E. C., HARRISON,R. J., AND HOBBS,J. R. 1968. Hyperproteinaemia GORDON-SMITH, associated with multiple myeloma. Proc. roy. SOC.Med., 61, 1112. P. F. 1973. Cryoimmunoglobulins. Sem. Hematol., 10, 87. GREY,H. M., AND KOHLER, HAYAT,M. A. 1969. Uranyl acetate as a stain and fixative for heart tissue. Proc. 27th Ann. Meet. Electron Micros. SOC.Am., Baton Rouge, p. 412. HIRSCH,J. G., AND FEDORKO, M. E. 1968. Ultrastructure of human leukocytes after simultaneous fixation in glutaraldehyde and osmium tetroxide and " post fixation " in uranyl acetate. J. Cell. Biol., 38, 615. HORNE,R. W. 1971. Electron microscopy applied to the study of macromolecular components assembled to form complex biological structures. symp. SOC.Exp. Biol., 25,71.
WHITE,ADAM,LAU,HORNE AND PARKHOUSE CRYOIMMUNOGLOBULIN IN PYODERMA
FIG.1.-Arterioles
PLATEVI GANGRENOSUM
in the dermis distended by protein, adjacent to area of purulent infiltration. Phosphotungstic acid haematoxylin (PTAH). x 480.
albumin IgG IgMs IgM FIG.2.-Analysis of acrylamide gel electrophoresis. ( a ) whole serum containing the cryoglobulin, (b) the isolated cryoglobulin. The position of marker proteins run in parallel is indicated on the figure.
FIG.3.-Tmmunoelectrophoretic analysis. ( a ) whole serum containing the cryoglobulin, (b) the isolated cryoglobulin. The upper two troughs contain anti-(normal human serum), and the lower one anti-human Fc isolated from IgG. Cathode on the left.
WHITE,ADAM,LAU, HORNEAND PARKHOUSE
VII PLATE
CRYOIMMUNOGLOBULIN I N PYODERMAGANGRENOSUM
FIG.4.-Isoelectric focusing of the isolated cryoglobulin. (a) (above) the isolated cryoglobulin, ( h ) (below)a monoclonal mouse IgG2. isolated from the serum of mice bearing the plasma cell tuiiiour Adj P C 5 .
FIG.5.--Crystallites of cryoglobulin in serum fixed at 4°C in simultaneous glutaraldehyde-osmium tetroxide, showing mainly longitudinal section of tubules. x 53,000. FIGS.5-7 are from electron micrographs stained by lead citrate and uranyl acetate.
W H I T E , ADAM,LAU, HORNE AND
PARKHOUSE
PLATEVIII
CRYOIMMUNOGLOBULIN IN PYODERMA GANGRENOSUM
FIG.6.-Parallel tubular units seen in longitudinal section in cryoprecipitated serum.
FIG. 7.-Cross-section
x 160,000.
of hexagonally-packed tubular units in cryoprecipitated serum, showing indication of structural subunits in and between the walls, and non-electron-dense lumina. x 160,000.
WIIITE, A D A M ,
LAU,HORNEAND
PARKHOUSE
PLATEIX
CRYOIMMUNOGLOBULIN IN PYODERMA GANGRENOSUM
FIG.8.-Electron micrograph of negatively stained cryoprotein. The small particles (arrows '' A ") correspond to the subunits observed as isolated components or in random aggre$?tes. Many areas of the specimens examined contained linear arrays of the type indicated at " B . x 60,oqO. Insert: high-magnification region showing groups of subunits associated with cryoprotern. x 230,000.
WHITE,ADAM,LAU, HORNEAND PARKHOUSE
PLATE X
CRYOIMMUNOGLOBULIN I N PYODERMA GANGRENOSUM
FIG.9.-Cross-section of small vessel in the dermis of skin biopsy, showing the lumen distended by precipitated protein, in which organised structure is visible. x 16,800.
FIG.
higher magnification of vessel contents, showing masses of hexagonally-packed tubules in cross-section. x 53,000. FIGS.9-12 are from electron micrographs stained by lead citrate and uranyl acetate.
101-A
WHITE,
ADAM,LAU, HORNE AND PARKHOUSE
PLATEXI
CRYOIMMUNOGLOBULIN I N PYODERMA GANGRENOSUM
FIG.
FIG.
I I .-Section
exhibiting various orientations of intravascular protein tubular assemblies. x 53,000.
12.-Protein masses with tubular structure lying in extravascular tissue in the dermis.
X
53,000.
33
CR YOIMMUNOGLOBULIN IN PYODERMA GANGRENOSUM
HORNE,R. W., AND PASQUALI-RONCHETTI, I. P. 1974. A negative staining-carbon film technique for studying viruses in the electron microscope. I. Preparative procedures for examining icosahedral and filamentous viruses. J. Ultrastruct. Res., 41, 361. A. 1970. The in vivo behaviour of twenty-four strains HULL,R., HILLS,G. J., AND FLASKITT, of Alfalfa mosaic virus. Virology, 42, 753. c. L., AND VAINSHTEIN,B. K. 1967. Crystallization of catalase KISELEV, N. A,, SHPITZBERG, in the form of tubes with mono-molecular walls. J. Mol. Biol., 25, 433. H. H., AND OREILLY,R. A. 1970. The hyperviscosity MACKENZIE,M. R. FUDENBERG, syndrome. I. In IgG myeloma. The role of protein concentration and molecular shape. J . Clin. Invest., 49, 15. MOROZ,L. A., AND ROSE,B. 1971. In Immunological diseases, 2nd ed., edited by M. Samter, Boston, vol. 1, p. 459. PARKHOUSE, R. M. E., AND ASKONAS, B. A. 1969. Immunoglobulin M biosynthesis. Intracellular accumulation of 7 s sbunits. Biochem. J., 115, 163. PARKHOUSE, R. M. E., VIRELLA, G., AND DOURMASHKIN, R. R. 1971. Structural characterization of a human monoclonal IgA protein. Clin. exp. Immunol., 8, 581. SAHA,A., EDWARDS, M. A., SARGENT, A. U., AND ROSE,B. 1968. Mechanism of cryoprecipitation. I. Characteristics of a human cryoglobulin. Zmmunochemistry, 5 , 341. D. M., MAIN,R. A., BECK,J. S., AND ALBERT-RECHT, F. 1973. Studies on a THOMPSON, patient with leucocytoclastic vasculitis, ‘‘ pyoderma gangrenosum ” and paraproteinaemia. Br. J. Derrn., 88, 117. H. H., AND COGGESHALL, R. 1965. A simplified lead citrate stain for use in electron VENABLE, microscopy. J. Cell. Biol., 25, 407. WELLS,R. 1970. Medical intelligence: syndromes of hyperviscosity. New Engl. J. Med., 283, 183. WHITE,J. G., AND HEAGAN, B. 1970. Tubular polymers of normal human hemoglobin. Amer. J. Path., 59, 101. Z. A., AND EDWARDS,G. A. 1972. Nonmyelomatous monoclonal immunoZAWADZKI, globulinemia. Progr. Clin. Immunol., 1, 105.
J. PATH.-VOL.
120 (1976)
C
