Role of blood coagulation in acute inflammation

Biochemical Pharmacology, Supplement, pp. 205-219. Pergamon Press. 1968. Printed in Great Britain

ROLE OF BLOOD C O A G U L A T I O N IN ACUTE INFLAMMATION* MARION I. BARNHART Department of Physiology and Pharmacology, Wayne State University School of Medicine, Detroit, Michigan Abstract--The inflammatory process is a response to many different kinds of tissue injury. One event that may be common to many apparently unrelated irritants is the deposition of fibrin. Three experimental designs have considered the significance of fibrin in acute inflammation, but only the third will be discussed extensively (details of the other approaches have been published). In the first approach, the biochemical profile of proteins concerned in fibrin formation was defined quantitatively for inflammatory fluids obtained from acutely inflamed joints of humans with arthritic diseases. Quantitative differences appeared to reflect degrees of inflammation rather than to define a profile unique for the types of arthritis. The second experimental approach attempted to correlate the type of exudative leucocytes with the presence of fibrin in human joint disease and experimental skin windows on dogs. The granulocyte phase of inflammation was maintained and prolonged by the presence of fibrin. The third experimental approach was a quantitative study of leucocyte response to the chemotactic power of fibrin, fibrinogen, and their proteolysis products. By a skin window collection chamber technique with dogs, the leucocyte responses to fibrin-related material, other protein aggregates (albumin, y-globulin, antigen-antibody complexes), and several inert particulate or structured preparations were compared. Fibrin-related materials were the most potent chemotactic agents for granulocyte (predominantly neutrophil) emigration in acute inflammation. ACUTE i n f l a m m a t i o n is triggered b y a variety o f a p p a r e n t l y unrelated irritants. Some o f these agents m a y o p e r a t e via different m e c h a n i s m s b u t this does n o t rule o u t a c o m m o n m e c h a n i s m for m a n y o f the irritants t h a t p r o m o t e the acute i n f l a m m a t o r y sequences. T h e p r o p o s i t i o n t h a t fibrin d e p o s i t i o n m a y p l a y a central role is considered in this c o m m u n i c a t i o n . First, there are present in i n f l a m m a t o r y fluids various b l o o d c o a g u l a t i o n proteins, o p p o r t u n i t i e s for their activation, a n d evidence o f their utilization. Second, the types o f i n f l a m m a t o r y cells a t t r a c t e d to a n d persisting at the i n f l a m m a t o r y site correlate well with fibrin deposition. Finally, a quantitative study o f leucocytes, e n c o u r a g e d to m i g r a t e into collection chambers, reveals t h a t greater n u m b e r s are a t t r a c t e d by the presence o f fibrinogen-related molecules t h a n by other p l a s m a proteins. MATERIALS AND METHODS

It~ammatory fluids These were o b t a i n e d by sterile a s p i r a t i o n o f acutely inflamed j o i n t s o f patients with various types o f r h e u m a t i c disease. The patients were classified a c c o r d i n g to the diagnostic criteria o f the A m e r i c a n R h e u m a t i s m Association. Care was taken d u r i n g * These investigations were supported in part by grant HE-3447 from the National Institutes of Health, Public Health Service. 205

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MARION [. BARNHART

joint aspiration to avoid rupture of blood vessels so that synovial fluid would not be contaminated with blood. Coagulation was prevented by immediate addition of sodium citrate (3.8 ~o). Patient details and a further description of our methods of synovianalysis are published2 -4

Proteins and other materials for experimental lesions Purified fibrinogen was prepared in this laboratory by the tannic acid method", and by the method of Blomb~ick and Blomb~ick.6 Fibrin nets were obtained by treating purified fibrinogen with purified bovine thrombin (kindly supplied by Dr. W. H. Seegers). Also, fibrin nets were prepared aseptically from the dog's own blood by recalcification of the anticoagulated plasma. Fibrinogen proteolysis products, especially the ~2-fibrinogen derivative D, resulted from digestion of fibrinogen by fibrinolysin and separation of the digestion products by polyacrylamide gel filtration. 7 The purified canine albumin was 95 ~o pure; human albumin (crystallized, 100 per cent pure) came from Mann Research Lab. Inc., New York, N.Y. Canine 7-globulin (98~, Mann) and human 7S globulin (100~, Immunology Inc., Lombard, Ill. and Mann) were used. Heat denaturation (at 56 °, 66 °, or 92 °) of either albumin or 7globulin (10~) in saline provided the respective protein aggregates for comparison with fibrin. Antibodies against either albumin or fibrinogen were elicited in rabbits according to published methods. 8 Antigen-antibody complexes were prepared by interaction of the purified protein with its antibody in a saline environment or by reacting serum containing albumin with its antibody. Complexes with fibrinogen were centrifuged from the reaction mixture after 1 hr at 37°; albumin complexes were incubated overnight at 4 °. In some cases, antigen-antibody complexes formed in saline were resuspended in serum and incubated at 37 °, centrifuged, and used in the experimental chambers. Thus. antigen-antibody complexes were prepared with and without the opportunity for adsorption of serum opsonins. Glass beads (type 660-5005, 3M Reflective Products Division, St. Paul, Minn.) and graded porosity membranes (Gelman instrument Co., Ann Arbor, Mich.: Millipore Filter Corp., Bedford, Mass.) provided inert particles and structured matrixes for comparison with protein aggregates. Saline (0.9 ~) and distilled water were sterile and of low particle content (Sherman Laboratories, Detroit, Mich.). Animal experimentation The majority of experiments were made on healthy, unanesthetized, trained dogs. The skin window procedure does not cause the animal discomfort (it was constantly attended during the experiment and thoroughly enjoyed the attention). Several experiments were on dogs anesthetized with sodium pentobarbital. Anesthesia without other stresses did not reduce significantly the leucocyte emigration into the inflammatory site. Furthermore, as many as sixteen to eighteen separate lesions could be placed for comparison (Fig. 1). Skin window technique. This procedure, essentially that of Rebuck and associates, '~ permits collection of inflammatory cells on cover slips for later study. Our modifications of this procedure for use on a dog's back are published.1° This technique is especially valuable for characterizing the various leucocyte types present at different

Fl(;, 1. Placemenl of skin window collection c h a m b e r s on shaved back of dog. A colored adhesive h¢~lds the c h a m b e r s o~.el the inflammatory site. Two standard skin windov.s (co~.ered completely by tape) were placed for comparison,

Fl~;. 2. Assembly o f skin window collection c h a m b e r s . Pla,dic parts A, I], and C are combined with n l e m b r a n e lilters to form a t w o - c o m p a r t m e n t chamber. 1) shows the m e m b r a n e filler adherent to parl A and is placed nearest the experimental lesion. Fluid can be introduced or withdrawn from the chanlber. E shows the tipper side of part D without the top portion (A and B) of the chamber. F shows that Illc membrane filter D can be cut away, allowing the inflammatory fluid with its leucocytes to be withdu|wn. The separate paris with their adherent membranes are wrapped in aluminum 11~il for sterilization.

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Inflammation at the cellular level--lI

207

times during the inflammatory sequences. However, quantitation of the total number of cells at any one stage of inflammation is not possible. To achieve this objective we have made use of diffusion chambers to collect the various exudative cells responding to the applied stimulants. Skin window collection chambers. The collection chambers used in this study are illustrated with their respective parts and various stages of assembly (Fig. 2). Details of the construction and use of the diffusion chambers described by Shelton and Rice n were basic to our design. The Grafar Corp. (Detroit, Mich.) provides the acrylic Plexiglas parts for the collection chamber. Three clear plastic discs (dia. 32 mm) are required. Disc A is 2 mm thick and has an access hole (dia. 0-75 mm) to an inner cut-out area (dia. 15 mm). Disc B is 1.5 mm thick and has an open central area (dia. 15 mm). Disc C is 1.5 mm thick. Nontoxic Acryloid B-7 plastic glue (Rohm and Haas, Detroit, Mich.) was used to glue the acetate membrane filters of designated porosity to the plastic discs. A 0-1-/~ filter was glued to disc B. An 8 or 10/z filter was glued to disc C and will ultimately contact the skin lesion on the dog's back. When discs B and C are glued together, two membrane-limited chambers are formed; the bottom one is completely membrane enclosed while the upper chamber has its top fully open at this time. This assembled portion (Fig. 2, D) is completely dried overnight at room temperature. Sterilization of the assembly is accomplished at 80 ° (dry heat) for about 24 hr. Each chamber is individually wrapped in unwrinkled aluminum foil and several are placed in a Petri dish with a cover. Disc C is wrapped separately and several are placed in another Petri dish and sterilized as indicated. Seating and removal of collection chambers. The final preparations of the collection chambers are made just before the skin of the dog's back is abraided. The dog's back is prepared the previous day by washing, clipping, and applying Surgex hair remover (Crookes-Barnes Lab. Inc., Wayne, N.J.) and a final treatment with pHisoHex (Winthrop Lab., New York, N.Y.). The areas for the experimental lesions are selected and outlined with a marking pencil. The freshly prepared protein aggregate and 0.2 ml of saline are introduced onto the top filter (0.1/~). Disc C is glued to disc B and closes the upper chamber. Either Acryloid B-7 or melted paraffin can be used to rim the discs and provide a firm seal. The chamber is turned over so the 10/x filter is exposed. Saline (0.4 ml) is injected through the access hole into the central compartment (Fig. 2, D). The access hole is plugged with paraffin or wax. The rim of the membrane is coated with nontoxic liquid adhesive (Acryloid B-7 or M F cement, Millipore Corp. or V Drape Adhesive) which drys partially to permit prompt adhesion of the chamber to the skin when ready. An area dia. 20 mm) is scraped on the dog's back by using a sterile Bard-Parker blade and continuing until fine bleeding points are obvious over the measured area. The prepared chamber is firmly seated over the superficial wound and held briefly to ensure a good seal (Fig. 1). Preparation of the lesion and seating of the chamber over it requires about 1 min. For additional stability, the entire chamber and adjacent area of the back are sprayed with liquid adhesive (V Drape Adhesive or Aeroplast Dressing, Parke Davis and Co., Detroit, Mich.). Finally, adhesive tape (1 in.) may be criss-crossed over the chamber to hold it securely even when the dog is moving. After the desired time interval, the collection chamber is carefully removed from the dog's back. With the chamber setting on its plastic top, the visible membrane

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MARION I. BARNHART

(10t~) is cut out (Fig. 2, F). The fluid with inflammatory cells from the central chamber is carefully withdrawn into a syringe and measured. Leucocyte counts are made on the undiluted fluid and, when necessary, on dilutions of it. Smears are also made for later staining by Leishman's method and for differential counts. The small amount of fluid in the upper chamber is taken up in a syringe, measured, and smeared on slides for later study. The upper membrane (0"lt~) is removed from the chamber and marked to indicate the upper side. This filter is fixed in 70 ~ isopropyl alcohol. Later it is stained with hematoxylin and eosin for examination. Because the membrane is of too fine a porosity for leucocytes to pass, presence of leucocytes above it indicates a tear in the membrane. Leucocytes on the 0"lt~ filter or in the smears from the fluid in the upper chamber indicate a defective chamber. Usually the membrane and upper fluid are free of cells, which verifies the proper functioning of the central chamber and the reliability of the quantitative results. Care of anhnal after experimental lesions. When exposed to air, the lesion is immediately sealed by the dog's own transudated fluids. Later, the liquid adhesive around the lesions is stripped away and if necessary the back is gently washed. The animal is returned to its quarters. Over the next few days the lesions are carefully examined for any signs of infection. Usually healing is uneventful and a dog can be reused in 2-3 weeks if desired.

Blood coagulation proteins in il~ammatory fluids Synovial fluid normally contains little or no blood clotting proteins, r', lz Quantitative study of synovial fluids taken from selected patients with joint disease revealed the presence of two essential coagulation proteins, fibrinogen and prothrombin.", '~ This collaborative work with Drs. Bluhm and Riddle of Henry Ford Hospital now includes 230 specimens. Because considerable care was taken with the joint aspiration, it seems unlikely that the observed prothrombin and fibrinogen resulted from admixture of blood. Thus, these plasma proteins entered the synovial fluid during the inflammatory process. In the absence of activation mechanisms, prothrombin and fibrinogen maintain their identity. The concentrations varied (Table 1) but seemed TABLE 1. PROTEIN CONTENT OF INFLAMMATORYFLUIDS FROM JOINTS Blood clotting Proteins Fibrinogen (mg/ml) Fibrin* Fibrin split products (mg/ml) Prothrombin (units/ml) Antithrombint Cathepsin (units/ml)

Osteo

Synovial fluids from various types of arthritis Gouty Rheumatoid Septic

0.08-0.24 0

0 2.5 32

0-2'7 77

0.15-0.57 0

0.24).8 40-86 -4-120

0'3-5'6 39-177 7-36 5-120

0.2-6.4 0-180 0-31 3-180

1.2-7.2 55-134 -8-114

Misc. 0 1.74 0 0.4-7.2 0-156 0-27 3-193

* Percent of patient series with gross fibrin in aspirated fluid. t Expressed as percent thrombin destroyed in test system. Normal range is 15-50 per cent. related to the magnitude of the inflammatory response rather than to the type of joint disease. Fibrinogen-related molecules. Fibrinogen (clottable by thrombin) was found in more than 85 per cent of these synovial fluids. Values approximating plasma levels

Inflammation at the cellular level--II

209

(1"5-2.5 mg/ml) were occasionally found, but the majority ranged between 0.18 and 0.97 mg/ml. Also, fibrin that had formed h7 vivo was detected especially in the anticoagulated fluids from rheumatoid arthritic joints. Evidence of fibrinolysis was gained when immunochemical tests measured molecules related to fibrinogen but not clottable by thrombin. Values for fibrin degradation products varied between 0.2 and 7-2 mg/ml. Thus, increased/3-globulin in many of these synovial fluids represented fibrinogenrelated molecules. Excluding the synovial fluids from patients with osteoarthritis and several near-normal ones, more than 78 per cent of the fluids had fibrinogen-related /3-globulin in excess of the normal plasma value of 17 per cent. Procoagulants. Prothrombin (Table 1) was found in more than 85 per cent of the synovial fluids. Values ranged down from 180 Iowa units/ml, which is a reasonable plasma value. A procoagulant, cathepsin, from cells was also identified in these synovial fluids. 2, 4 Prothrombin can be activated to thrombin and autoprothrombin C when cathepsin is present with lipid. 14, 15 Cathepsin is a cellular enzyme system that has ability to digest albumin. 16 The albuminolytic power of these fluids varied between 3 and 193 units/ml (Table 1). This activity at pH 4 reflected cathepsin D activity. In some cases, cathepsin E was measurable. The cellular source of much of the cathepsin was the exudative neutrophil. Many of these cells had lost their specific granules (lysosomes) and the pattern of intracellular cathepsin distribution was altered from that in the peripheral blood neutrophils according to fluorescent immunocytology.1, 2, 17 Additional procoagulants have been reported recently by other laboratories. Factors V, VIII, and XII were reported in pathologic synovial fluids by Lewis and associates. 18 Factors XI and XII were found in normal synovial fluids by Kellermeyer and Breckenridge. 19 Lipids have long been known to be increased in synovial fluids from diseased joints. 2o, 21 Anticoagulants. These were identified in synovial fluids by our study group in Detroit. 3 Antithrombin was measured in 80 per cent of our series and neutralized from 7 to 36 per cent of the thrombin added to the test system. Some fibrin degradation products can exert an anticoagulant effect.2z, 23 Most of the synovial fluids of our series had significant amounts of fibrin degradation products (Table 1). Interrelationships. These results illustrate that, during inflammation and dependent on its degree, the potential for fibrin formation, as that for anticoagulation, exists or develops in synovial fluids. The balance between these procoagulant and anticoagulant tbrces determines whether or not fibrin deposition is the end result. In rheumatoid arthritis and gout the procoagulant forces had obviously excelled because fibrin was visible as masses and strands in many of the synovial fluids. Exact identification of these deposits was gained with the aid of fluorescent antifibrinogen. 17 Furthermore, the low prothrombin concentration in many synovial fluids from patients with rheumatoid arthritis was not in accord with the evidence of enhanced transudation of other globulins. 3 Thus fibrinogen-fibrin transformation was promoted within the joint in response to local activation.

Correlation of granulocyte emigration with fibrin deposits In the response to tissue injury, capillary changes occur that favor the movement of leucocytes from intact blood vessels. Both the magnitude of this reaction and the nature of the extravascular attractants vary with the injury. While the cellular

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MARION I. BARNHART

scquences of the inflammatory process are somewhat stereotyped, the controlling mechanisms continue to mystify. Leucocytes in pathologic synovial fluids. Leucocytes are relatively rare in normal synovial fluid but increase in number during the exacerbations of joint disease. 12 Also, there is a shift away from mononuclear cells as the predominant cell type in normal synovial fluid. Polymorphonuclear cells become numerous in joint fluids from patients with gouty arthritis, infectious arthritis, or rheumatoid arthritis. 1~ Our findings are confirmatory. Attempts to correlate total leucocyte count with various proteins known to exist m these inflammatory exudates draw attention to fibrin#, 3 There was no correlation between leucocyte count and the synovial fluid concentrations of albumin, prothrombin, profibrinolysin, fibrinogen, fibrin split products, or those molecules responsible for the differential rheumatoid factor titer. There was a positive correlation between leucocyte count and the presence of fibrin: of the fluids with detectable fibrin, 96 per cent had leucocyte counts exceeding 10,000/mm~. Furthermore, the absolute neutrophil counts of synovial fluids from patients with rheumatoid arthritis correlated with the in rico formed fibrin. 1 A mean of 15,760 neutrophils was found when fibrin was present while the mean without fibrin was 7400. It is recognized that some other particulates, such as antigen-antibody complexes, may attract the leucocytes. Such complexes may exist in the synovial fluids of patients with rheumatoid arthritis. But such a correlation with 7-globulin aggregates has not yet been demonstrated while the correlation of leucocytes with fibrin merits further consideration. Leucocytes in skin windows. Borrel124 in 1893 was the first to stress the cytological sequence of inflammatory cells, with polymorphonuclear cells appearing earliest. Menkin z5 and m a n y other investigators have confirmed this observation. Rebuck and Crowley 9 showed a similar sequence with their skin window technique which we have applied to dogs (Fig. 3). lc"°r!

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Inflammation at the cellular level--II

211

Experimental inflammation in dogs 1°, 26 and in humans 27--29 documents the importance of fibrin for the granulocytic phase of inflammation. Various purified proteins that may gain entry to an inflammatory site were tested, in sterile skin windows in healthy dogs, for their effect on the pattern of leucocyte emigration (Fig. 3). Fibrinogen, fibrin, and proteolytic enzymes that elicit fibrin formation or degradation (thrombin, trypsin, and cathepsin) promoted an early emigration of eosinophils with peak responses at 4-6 hrY 0 As much as 20-50 per cent of the cell population was eosinophils (Fig. 4). Neutrophils also were present during this time (Fig. 3). Mononuclear cells (hypertrophied lymphocytes, lymphocytes, monocytes, and macrophages) appeared in PER CENT EOSINOPHILS 0

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FIG. 4. Selective attraction of eosinophils into an inflammatory site. D. T. ~ diphtheria toxoid which serves as a nonpyrogenic control; saline produces a similar control response. A differential count of 500 exudative cells was made for each stimulant. This dog had 8 per cent of its peripheral blood leucocytes as eosinophils. increasing numbers after 4 hr and later on were the predominant cell type. Repeated application of sterile fibrin nets from the dog's own blood prolonged the granulocytic phase of inflammation. The continued presence of fibrin for 14 hr kept the leucocyte population 100% granulocytic with neutrophils predominant and exclusive from 8 to 14 hr. 26 Neither albumin nor histamine evoked the described eosinophilic response. Immunofluorescent studies showed that eosinophils released profibrinolysin to the inflammatory fluid 1° while neutrophils phagocytized and digested fibrin. 26 Confirmation of the ingestion of fibrin by neutrophils was gained by ultramicroscopy. % Similar results have been obtained in studies of human skin windows exposed to sterile, autologous fibrin nets. 27 The cytologic features of such sterile inflammations were followed over 24 hr; neutrophils were the predominant cell type as long as sufficient fibrin was present in the lesion. About 80 per cent of the leucocytes were neutrophils from 9 hr on, in contrast to control lesions which had 50 per cent by 14hr. 28 An interesting study by Rebuck and collegues 29 on allergic patients showed that fibrin was associated with the profuse eosinophil response to pollen grains introduced into skin windows. The granulocytic response persisted for the duration of the study, 48-72 hr. The nonallergic patient showed only a few fibrin strands and the early reaction was predominantly neutrophilic, followed by the typical mononuclear pattern later. In skin windows placed on patients with nonspecific chronic uveitis, application of uveal pigments induced fibrin deposits and a tremendous outpouring of eosinophils

212

MARION [. BARNHART

As early as 1 hr after introduction of insulin into the skin window of insulin-resistant diabetic patients, a similar association of fibrin with eosinophil predominance was observed. Studies such as these certainly encourage the idea that fibrin or its degradation products behave as specific attractants for granulocytes in acute inflammation.

Chemotactic power of fibrh7 in skin window collection chambers Because the described skin window work was semiquantitative, attempts were made to improve the experimental design to determine whether or not the granulocyte emigration was related specifically to fibrin or was just a response to particulate material or organized structure. It seemed reasonable that diffusion chambers n might aid collection of leucocytes from the abraded skin surface and permit in vi~,o experimentation. This proved to be a valuable method for the quantitation to be described later. Leucocyte penetration of graded-pore membrane filters. I t was known that 0. l/z anti 0.45/z m e m b r a n e filters were impervious to cells when these membranes were in diffusion chambers placed in the peritoneal cavity. 3°, at However, the smallest porosity for leucocyte penetration was not witb out dispute: Algire 31 reported cell penetration with o'8tz filters while Gregoire 3') stated that 1 to 3/z membranes were impervious to cells. We decided to interpolate various sizes of membrane filters (0.01-10'0tz) between the scarified skin and the collecting cover slip in our standard skin window procedure. To avoid m o v e m e n t of the leucocytes around the edges of the filter rather than through its pores a piece o f tape was placed over the filter so that movement was restricted to the area in contact with the cover glass. Sixty-four skin windows were collected with the improved technique over 15 hr and were evaluated. Comparison was made between saline controls and responses to fibrin nets placed on the cover slip (Table 2). Membranes up to 0.45/z were impervious TABLE 2. LEUCOCYTE PENETRATION OF GRADED PORE MEMBRANES DURING ACUTE INFLAMMATION HF

Filter Porosity (p.) Stimulus 0.05 0.10 0.30 0.45 0.65 0.80 5-0 I0.0

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to leucocytes even when fibrin was present as a stimulant. However, leucocytes were capable of penetrating membranes of 0"65tz or greater. Leucocytes penetrated the 0"65 and 0.80/x filters more rapidly when fibrin was on the cover slip. This was especially evident in the experiments with 0.65tz filters: neutrophils were obvious at

Inflammation at the cellular level--It

213

2 hr in the presence of fibrin in contrast to saline controls requiring 15 hr before leucocytes were detected. Many of these polymorphonuclear cells fragmented as they passed through the narrow pores to reach the fibrin. Because the minimal nuclear lobe width is near 0.5/,, it is not surprising that portions of the cytoplasm were separated from the main body during the 150-/, journey through the 0.65-/* pore. With membranes of 5- to 10-/z pore size, leucocytes readily traversed the pores and intact ceils were collected on the cover slip. Time study of leucocyte emigration to fibrin. The knowledge gained on membrane penetration was applied in the construction of the diffusion chambers (Fig. 2). The first collection chambers were charged with fibrin nets prepared from dog's own anticoagulated blood by recalcification. These fibrin nets rested on the moist upper filter (0.l/z) with a saline continuum to the test lesion. Any leucocytes that passed the wide pore filter (8-10/*) were in the inner collecting chamber and could not pass the narrow upper filter to adhere to the fibrin and be lost. A collecting chamber was removed for leucocyte counts at 2, 4, 6, 8, and 9 hr (Fig. 5). The leucocyte count increased from

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1080 to 4810/mm. a Granulocytes comprised 92-100 per cent of the cell population during the test period. From saline controls taken at 4 and 8 hr, fewer leucocytes were collected and counts were about 10 per cent of those from comparable fibrin windows. Thus, we could get a reasonable count of ceils responding to a chemotactic stimulus within 4-6 hr. Leucocyte response to coated and uncoated beads. Another experiment tested whether the leucocyte emigration into the collecting chambers was a response to structure or to particles per se. For this study, fine glass beads (44/,) were carefully washed and placed in the upper compartment of the collecting chamber. Other collecting chambers contained beads coated with purified canine fibrinogen. A fibrinogen and a saline

214

MARION I. BARNHART

control completed this series of collecting chambers which contacted the skin windows for 4 hr. More leucocytes were attracted by the fibrinogen-coated beads and by the fibrinogen than by saline or bead controls (Fig. 6). The beads coated with fibrinogen attracted two to five times as many leucocytes as did the saline or bead controls. Because the uncoated beads were essentially without effect in the collecting chambers, it seems apparent that the film of fibrinogen conferred chemotactic power on these beads•

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Leucocyte responses to heat-aggregated protein• The next experiments attempted to answer the question of whether the described neutrophil emigration to a fibrin stimulus was specific or was just a response to aggregated protein or structured masses• Comparison was made of the chemotactic powers of fibrin and heat-denatured and aggregated albumin and 7-globulin (Fig. 7). Neither albumin not" ~-globulin of canine or human origin elicited a greater migration of neutrophils than the saline control• In sharp contrast, fibrin called forth 6300 leucocytes/mm 3 as compared to counts of 550-675 achieved with the other agents• Clearly, fibrin was a more powerful chemotactic agent than the other aggregated proteins• Leucocyte responses to aged fibrin. In other experiments, fresh fibrin and aged fibrin were compared with controls• Precautions were taken to keep the fibrin nets sterile as they aged for several days. The saline controls varied between 475 and 725 leucocytes/ m m 3. Fresh fibrin and 1-day-old fibrin attracted from 1000 to 1500 leucocytes/mm3• Fibrin 2 and 3 days old did not elicit any more leucocytes than the saline controls• Apparently, aging the fibrin longer than I day destroyed its ability to stimulate leucocyte emigration. Either certain important constituents were released from the fibrin during its aging or the fibrin became stabilized and resistant to the proteolytic action

lnflamnmtion at the celhdar level--If

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required at the site of inflammation to release chemical messengers for attraction of cells. Leucocyte responses to antigen-antibody systems. Another approach involved comparison of the chemotactic power of two kinds of antigen-antibody complexes. An albumin system was prepared by interacting canine albumin with anti-albumin. The second, a fibrinogen system, was prepared by reacting canine fibrinogen with antifibrinogen. These complexes were prepared in saline and in serum environments and were placed in collecting chambers. Controls were saline and heat-aggregated albumin. The leucocyte emigration to the fibrinogen-anti-fibrinogen complexes was at least three times that elicited by the albumin-anti-albumin systems, heat-aggregated albumin, or saline (Fig. 8). There was little difference in the chemotactic power of albumin-antialbumin prepared in fresh serum or in saline. Thus, an opsonin effect was not observed. The most significant stimulus for leucocyte emigration into the chamber was the presence of fibrinogen, in this case held in complex to its specific ),-globulin antibody. These experiments and those with heat-aggregated y-globulin did not reveal any special chemotactic power of )'-globulin. Leucocyte responses to.fibrinolytic products. An attempt was made to determine what endowed the fibrinogen-fibrin system with its power to mobilize granulocytes into the skin window collecting chambers. In this test system it seems unlikely that the stimulus was just organized structure, including the fibrin itself, because a cell-impenetrable membrane separated the fibrin from the inflammatory cells. Most likely, a chemical messenger was released from the fibrin to establish a concentration gradient which led the leucocyte into the collecting chamber. Some of the early work made use of mixed polypeptide fractions from hydrolysates of fibrin.33-3~ These fractions increased capillary permeability and leucocyte emigration.

216

MARION I. BARNHART

Various plasma protein fractions influenced leucocyte migration in v i t r o . "~6 Fibrinogen has been injected and induced inflammation with attendent leucocyte emigration. 37 However, the precise nature of the stimulants was difficult or impossible to establish at that time.

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ControlAlbumi AlnbumiAlnbumiFinbrinogen HEAT' ANTIGEN AGG. ANTIBODY COMPLEXES FiG. 8. Leucocyte responses to heat-aggregated a l b u m i n a n d two antigen-antibody systems. The a l b u m i n system on left was prepared in saline while the a l b u m i n system on right was prepared in serum. T h e control collection c h a m b e r h a d saline added.

In recent years, several derivatives or proteolysis products of fibrinogen and librin have been isolated and characterized. One or more of these may be responsible for the described effects on inflammation. As fibrinogen is transformed to fibrin, fibrinopeptides and fibrin monomer are released. There is already evidence from the work of Copley and associates 3s that fibrinopeptides A and B increase vascular permeability. To my knowledge, neither fibrinopeptides nor fibrin monomer have been tested for a leucotactic ability. Such experiments are necessary for a full understanding of the role of fibrinogen-fibrin in inflammation. Two other fibrinogen derivatives (D and E) have been recognized as degradation products of the enzymatic breakdown of either fibrinogen or fibrin. The major proteolysis product of either fibrin, fibrin monomer, or fibrinogen is fibrinogen derivative D which is a B-globulin and is characterized immunologically by possession of the antigenic determinant group D of Nussenzweig and Seligmann. a:) Fibrinogen derivative D, purified from a canine fibrinogen-fibrinolysin digest, was tested for its leucotactic ability in skin window collecting chambers (Fig. 9). Two concentrations (0.5 and 2.0 mg/ml) were compared with fibrinogen at 2 and 4 mg/ml

Inflammation at the cellular level--ll

217

and with a saline control. Fibrinogen derivative D was more powerful than fibrinogen in eliciting leucocyte accumulation. As proteolytic enzymes, transudated from blood or released from injured or functioning cells, become constituents of inflammatory fluid, the emergence of fibrinolytic products is not unreasonable. Fibrinogen split products have been measured in one

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Leucotactic ability of purified fibrinogen and its proteolysis product, fibrinogen derivative D. The control lesion had saline added.

type ofinfiammatory fluid, pathologic synovial fluids. 3 Now it has been shown that one of the fibrinolytic products, fibrinogen derivative D, stimulates leucocyte emigration and accumulation. This fibrinolytic product is four to eight times as effective as the parent molecule, fibrinogen. On the basis of this quantitative work, fibrinogen derivative D is a good candidate for the role of chemical messenger encouraging leucocyte emigration during inflammation. CONCLUD[NG REMARKS Although Opie 40 near the turn of this century and Menkin 25 and Jansco 41 more recently called attention to the association of fibrin, leucocytes, and inflammation, this relationship has not been understood. Our results make it seem unlikely that the association is just coincidental. Obviously, multiple mechanisms must operate in the exquisitely timed sequences of the inflammatory process. However, there is no reason to turn away from a unifying concept that may relate some of the irritants and further explain the persistence of acute inflammation under conditions of continued fibrin deposition. The existence of fibrin and fibrin promoting agents in inflammatory fluid, the demonstration of a selective stimulation for granulocyte emigration in experimental sterile inflammation, and finally a quantitative consideration of the relative powers of various proteins as chemotactic agents for granulocyte accumulation offer support for the concept that fibrin and its immunologic relatives play a central role

218

MAR1ON I. BARNEIART

i n a c u t e i n f l a m m a t i o n . T h e c a s e f o r fibrin is b y n o m e a n s c l o s e d : r a t h e r , it h a s j u s t been reopened. Acknowledgements--The technical assistance of Mrs. Arlys Vettraino, Miss Sharon Agree, G. W. Moore, and Len Sulisz was appreciated. REFERENCES 1. 2. 3. 4.

J. M. RIDDLE, G. B. BLUHMand M. 1. BARNHART,J. Retieuloendothelial Soc. 2, 420 (1965). G. B, BLUHM, J. M. RIDDL~ and M. 1. BARNHART, Henry~FordHosp. med. Bull. 14, 119 (1966). M. 1. BARNHART,J. M. R1DDLE, G. B. BLUHMand C. QUINTANA,Ann. rheum. Dis. 26, 206 (1967). M. 1. BARNHART,C. QUINTANA, H. L. LENON, G. B. BLUHM and J. M. RIDDLE, Ann. N. Y. Acad. Sci. in press. 5. M. I, BARNHART and W. B. FORMAN, in Blood Coagulation, Hemorrhage and Thrombosis (Eds. L. TOCANTINS and L. KAZAL). Grune & Stratton, New York (1964). 6. B. BLOMBT,CK and M. BLOMB,~CK,Ark. Kemi 10, 415 (1956). 7. M. [. BARNHART, D. C. CRESS, R. L. HENRY and J. M. RIDDLE, Thromb. Diathes. haemorrh. 17, 78 (1967). 8. M. I. BARNEIART,G, F. ANDERSON and W. BAKER, Thromb. Diathes. haemorrh. 8, 21 (1962). 9. J. W. REBUCK and J. H. CROWLEY, Ann. N.Y. Aead. Sci. 59, 757 (1955). 10. J. M. RIDDLE and M. I. BARNHART, Blood25, 776 (1965). 11. E. SHELTON and M. E. RICE, J. natn. Cancer Inst. 21, 137 (1958). 12. M. W. ROPES and W. BAUER, Synorial Fluid Chan,~,es in Joint Disease. Harvard University Press, Cambridge (1953). 13. M. H. CHO and O. W. NEUHAUS, Thromb. Diathes. haemorrh. 5, 108 (1960). 14. G. M. PURCELLand M. I. BARNHART, Bioehim. biophy~. Acta 78, 800 (1963). 15. G. M. PURCELLand M. 1. BARNHART, Physiologist 7, 229 (1964). 16. C. LAPRESLEand T. WEBB, Bioehem. J. 76, 538 (1960). 17. M. I, BARNIa'ART,J. M. RIDDLE and G. B. BLUHM, Ann. rheum. Dis. 26, 281 (1967). 18. J. H. LEWIS, [. L. SZETO, R. C. MARTINEZ, W. H. BAYER,J, A. SPEROand G. P. RODNAN, Arthritis Rheum. 9, 520 (1960). 19. R. W. KELLERMEVERand R. T. BRECKFNR1DGE,J. Lab. clin. Med. 67, 455 (1966). 20. D. H. KLING, The Synovial Membrane And The Synot~ial Fhtid. Medical Press, Loa Angeles (1938). 21. G. C. BOLE, Arthritis Rheum. 5, 589 (1962). 22. J. HIRSH, A. P. FLETCHER and S. SHERRY, Am. J. Physiol. 209, 415 (1965). 23. D. C. TRIANTAPHYLLOPOULOS, Can. J. Biochem. Physiol. 36, 249 (1958). 24. A. BORRELL, Ann. hist. Pasteur 7, 593 (1893). 25. V. MENKtN, Biochemical Mechanism., In hzflammation. Charles C. Thomas, Springfield (1956). 26. J. M. RIDDLE and M. I. BARNHART, Am. J. Path. 45, 805 (1964). 27. J. M. RIDDLE, N. A. ODLE, G. B. BLUHM and M. 1. BARNHART, Thrombo3. Diathes. haemorrh. 18, 302 (1967). 28. N. A. ODLE, A Study Q f The ht[tammatory Response ht Patients With RheumatoM Arthritis. Master of Science Thesis, Wayne State University, Detroit (1966). 29. J. W. REBUCK, L. C. SWEET and C. L. BAP,TH, Fedn Proe. 26, 745 (1967). 30. E. SttELTON, J. Cell Biol. 12, 652 (1962). 31. G. H. ALG~RE, Ann. N. Y. Acad. Sci. 69, 663 (1957). 32. C. GREGOIRE, Q. JI. mierosc. Sci. 99, 511 (1958). 33. E. S. DUTHIE and E. CHAIN, Br. J. exp. Path. 20, 417 (1939). 34. W. G. SPECTOR, J. Path. Bact. 63, 93 (1951). 35. G. A. TERSHAKOVECand V. H. MOON, Am. J. Path. 28, 522 (1952). 36. M. M. KETCHELand C. B. FAVOUR,J. exp. Med. 101,647 (1955). 37. R. A. PAZ and W. G. SPECTOR, J. Path. Bact. 84, 85 (1962). 38. A. L. COPLEY, J. P. HANIG, B. LUCHINI and R. L. ALLEN, JR., Fedn Proc. 25, 446 (1966). 39. V. NUSSENZWHG and M. SELIGMANN,Retd. Hemat. 15, 452 (1960). 40. E. L. OP~E, J. exp. Med. 9, 391 (1907). 41. N. JANCSO,J. Pharm. Pharmac. 13, 577 (1961).

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COMMENTS In response to a question by Dr. Ward concerning the lack of chemotactic action of the serum as found in the in vitro chamber system of Boyden, which he had employed, Dr. Barnhart stated that with clot formation and its subsequent lysis, the amount of immunologically related fibrinogen derivatives that can be measured increases. Thus normal serum contains 0.2-0.4 mg of fibrinogen degradation products, perhaps normally being thrown off as a consequence of fibrinolysis. If clot formation is followed by lysis, this serum fibrinogen-related material, which may be characterized by the fibrinogen-derivative D, reaches a magnitude of 1.0-2.0 mg. Thus chemotaxis may be a concentration dependent action. Dr. Ward then added that in recent work in his system, if the plasmin system was activated, he obtained an additional factor, a split-product (C'3) which was chemotactic for leukocytes. In reply to a question of Dr. Barnett's as to whether dogs had been intentionally sensitized to fibrin and then studied, Dr. Barnhart stated that two of the dogs to which fibrin had been applied in skin windows at intervals for 5 yr still had not developed anti-fibrin antibody. But she and her colleagues had found a circulating anti-fibrin antibody in a h u m a n patient suffering from thrombophlebitis migrans and the appearance and disappearance of the antibody coincided with the presence and absence of an active inflammatory state. She had not actually intentionally sensitized dogs with fibrin in adjuvant. Finally in reply to a question of Dr. Willoughby's as to whether the activity of fibrin in this work might have been related to its inability to move away from the inflammatory site, Dr. Barnhart pointed out that in the controls cited above, she had actually employed heat-aggregated albumin and y-globulin so that the effects of the structured fibrin could be more closely compared with the effects of other structured proteins, which proved relatively ineffective as chemotactic agents.

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