The Skin Window as a Monitor of Leukocytic Functions in Contact Activation Factor Deficiencies in Man

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1 The Skin Window as a Monitor of Leukocytic Functions in Contact Activation Factor Deficiencies in Man JOHN W. REBUCK, M.D., PH.D. I AM DEEPLY HONORED to be the recipient of the H. P. Smith Memorial Award of our Society. I had the privilege of knowing Dr. Smith as we labored in our differing fields of hematology. It was Dr. Smith and his equally accomplished colleagues and students who developed the quantitative titration procedures that gave us the methods for purifying the various clotting factors, the practical basis today for the diagnosis and treatment of hemorrhage and thrombosis in our patients. For this reason my remarks will center on an area where our respective fields of interest, coagulation and leukocytic functioning, have merged: a merger involving the contact activation system. The contact activation system is composed of Factor XII or Hageman Factor, 24 prekallikrein (PK) or Fletcher Factor, 13 and High Molecular Weight Kininogen (HM r K) or Fitzgerald Factor, 6 ' ' 39 the eponymic designations following those of their propositi. Their discoveries were made in the listed sequence; historically, each manifested a lack of procoagulant activity when the plasmas of their propositi were exposed to the negatively charged surfaces of glass test tubes. The identity of each was established by the ability of plasmas deficient in an individual factor to correct the procoagulant deficiencies of each of the others in turn. In the coagulation scheme, activation of the contact system leads to activation of Factor XI in the intrinsic clotting system and of Factor VII in the extrinsic clotting system. 3 The observations that patients with plasma deficiencies of each of the respective contact activation factors suffered from no episodes of abnormal bleeding directed attention to their possible roles as chemical messengers in inflammation, chemotaxis and fibrinolysis. 2 ' 3 ' ' 23 ' ' 45 The contact activation system is brought into play by HM r K binding to the negatively charged surface with HM r K or part of it followed by HM r K-PK and HM r K- Factor XI complexes interacting with Hageman factor Received and accepted for publication November 1, Presented at the Joint Fall Meeting of the American Society of Clinical Pathologists and College of American Pathologists, October 1982, Miami Beach, Florida. Address reprint requests to Dr. Rebuck: Department of Pathology, The Henry Ford Hospital, 2799 W. Grand Boulevard, Detroit, Michigan Department of Pathology, The Henry Ford Hospital, Detroit, Michigan similarly binding, 9 ' 32 since activation of Factor XI occurs only on the surface. HM r K is complexed with PK and Factor xi 817 ' ' 43 in the fluid phase of plasma and thus is in a position to carry these proteins to negatively charged surfaces. Once the surface is attained, the interaction between HM r K, Factor XII, PK, and Factor XI activates all the Factor XH-dependent pathways, 45 such as the complement system, kinin generation, fibrinolysis, kallikrein, and leukocytic chemotaxis in addition to intrinsic coagulation, all participants in the inflammatory response. At the site of injury in vivo, the negatively charged surfaces that could lead to HM r K binding are, variously, collagen, elastic tissue, skin, and basement membrane As early as 1965, Ratnoff and his group 10 had found that activated Factor XII produced a delayed but prolonged inflammation in the rabbit ear chamber. This inflammatory reaction was marked by margination or sticking of the leukocytes to the endothelium, followed by migration of the leukocytes into the tissues. In 1972, and again in 1974, Kaplan and his co-workers 1342 found that activation of PK to kallikrein was strongly productive of human granulocytic chemotaxis in the Boyden chamber in vitro. Conversely, PK deficiency resulted in a diminished chemotactic activity for human granulocytes in vitro. Kallikrein, however, recently has been shown to be chemotactic for leukocytes only if a source for C5 is available. 44 Since patients with Factor XII or PK deficiency had, as yet, no reported overt clinical evidence of depressed cell-mediated immunity, the patients themselves, deficient in such factors, had not been tested for possible leukocytic faults until our report of excessive leukocytic migration observed in the skin windows of a patient with Fitzgerald factor deficiency, 37 a deficiency soon identified as HM r K deficiency. In the following year, Hathaway and Wuepper and their colleagues, 12 using a modified skin window technic in which a plastic chamber was applied to the lesion, reported that the migration of PK deficient patients' leukocytes /83/0400/0405 $01.25 American Society of Clinical Pathologists 405

2 406 REBUCK. A.J.C.P. April 1983 surface of the body, the customary site being the volar surface of the forearm. The area selected is first shaved, if required, then cleansed with 70% alcohol. Longer lasting and more effective preparatory agents were avoided to prevent cytotoxicity of the exudative leukocytes. Two methods of monitoring leukocytic migrations were employed in the present study. The response to the trauma of the technic itself was followed in the first study (Figs. 1-8). The lesion was elicited by scraping the selected site with the tip of a scimitar-shaped scalpel (Bard-Parker #22) to a shallow saucer-like field which revealed minute bleeding points, the traumatized dermal papillary capillary networks. In as much as the epithelial rete pegs were left intact, the lesions healed without scarring. The time of abrasion was taken as the zero hour of the inflammatory response due to trauma alone, and a small round sterile coverslip was placed over the lesion, topped with a small square of sterile cardboard to lessen breakage, and surmounted with hypoallergic surgical tape. At 6 hours, the coverslip was removed, air-dried, and stained like a blood smear. A second coverslip was applied similarly and kept in place until the 24th hour of traumatic inflammation had supervened. John R. Rebuck, M.D., PH.D. to attractants at 24 hours was the same as control leukocytes. The latter findings apparently were the opposite of the earlier in vitro results of Kaplan and his colleagues.' 342 To provide morphologic documentation of the effects of deletion of each of the individual factors that make up the contact activation system upon the inflammatory response in man, human skin window tests were applied to patients with Factor Xll, PK, and HM r K deficiencies. Since PK deficient plasma after prolonged incubation paradoxically undergoes apparent restoration of ail its normal functions except for kinin generation, the inflammatory response in the entire group was studied early, at 6 hours of inflammation, and late, at 24 hours of inflammation. Furthermore, the leukocytic sequences were elicited after the trauma of the technic alone (Cohnheim's inflammation) and after application of an antigenic stimulus (MetschnikofFs inflammation). In a second study, cell-mediated immunity was monitored by adding an antigenic stimulus (0.05 ml diphtheria-tetanus toxoid) to the surface of the abrasion before mounting the first coverslip. The additional stimulus employed thus contained antigens for which there was known patient competency due to prior exposure. The responding exudate was again recovered at 6 and 24 hours (Figs. 9-12) of antigehically induced inflammation added to that produced by the trauma of the technic. In addition to adult male and female controls (Figs. 1, 2, and 9), six patients with contact activation deficiencies were studied as described with respect to their leukocytic response to the trauma of the technic alone and to the superimposition of an antigenic stimulus. The patient groups were as follows: three patients with Factor XII (Hageman factor) deficiency, a mother and her two young daughters* (Figs. 3, 4, and 10); two of the propositi of prekallikrein (Fletcher factor) deficiency (Figs. 5, 6, and 11); and the propositus of high molecular weight kininogen (HM r K) deficiency (Fitzgerald factor deficiency) (Figs. 7, 8, 12, and 13). The latter patient was restudied at a later interval because of the unusual nature of his response, and the study was continued until the 49th hour of inflammation (Fig. 14). Materials and Methods Skin window test lesions have been described in detail in prior reports. 24 " 26,28 The method consists of abrasion of a circle of skin exactly 3 mm in diameter on a plane * 1 am indebted to Dr. John Penner. Michigan State University for permission to study these patients, whose Factor XII deficiency he had established, and to Drs. R. Waldmann and J. P. Abraham of the Henry Ford Hospital for permission to study their patients with PK and HM r K deficiency.

3 Vol. 79. No. 4 H. P. SMITH AWARD LECTURE FIG. 1 {upper, left). Six hours inflammation due to trauma in test lesion in normal control. Low-power view for quantitation, for comparison with Figures 3, 5, and 7. Leishman's stain (XI15). FIG. 2 {upper, right). Six hours inflammatory response due to trauma alone, higher power of Figure 1, normal control. Note neutrophils and occasional monocyte. Leishman's stain (XI.000). FIG. 3 {lower, left). Six hours intlainmation due to trauma alone in a Factor Xll-deficient female child. Responding leukocyte numbers are only 4% of control numbers in Figure 2. Leishman's stain (XI15). FIG. 4 {lower, right). Higher power of a portion of the response shown in Figure 3 in Factor Xll-deficient patient. The few responding leukocytes are neutrophils and monocytes. Leishman's stain (XI.000). 407

4 % 40 */ * *j».>* to *». A3L «v,*»* FIG. 5 (upper, left). Six hours inflammation due to trauma alone in young male propositus of prekallikrein deficiency. This response is 4X greater than in the Factor Xll-deficient patient shown in Figure 3 but less than 20% of the normal quantity shown in Figure 1. Leishman's stain (XI15). FIG. 6 (upper, right). Higher power of a portion of the response shown in Figure 5 in a prekallikrein-deficient patient. The responding leukocytes are neutrophils and monocytes as shown. Leishman's stain (X1,000). FIG. 7 (lower, left). Propositus of HM,K deficiency (Fitzgerald factor). Six hours of inflammation due to trauma of the test alone. Excessive leukocytic exudate covers entire test lesion site with a fourfold increase over normal response (see Fig. I) but more than 100X the response in Factor XII deficiency (Fig. 3) and more than 20X that in PK deficiency (Fig. 5). Low-power view. Leishman's stain (XI15). FlG. 8 (lower, right). Same lesion as Figure 7 but higher magnification. Six hours of inflammation due to trauma alone in HMrK-deficient patient. Excessive leukocytic migration consists of neutrophils, eosinophils, basophils, monocytes, and lymphocytes. Leishman's stain (X 1,000).

5 vol. 79.No.4 H. P. SMITH AWARD LECTURE 409 Results Unlike the full exudate that covers the entire site of the lesion in response to stimulation by an antigen, for which the healthy volunteer is immunologically competent through prior experience with the antigen employed, the response to the trauma imposed by the technic alone in skin window preparation covered only one-fourth of the field covering the test lesion at the 6th hour (Fig. 1). This modest leukocytic exudate was composed of neutrophils, which had not degranulated, and a few accompanying monocytes (Fig. 2). In sharp contrast, in the three Fact XII (Hageman factor) deficient patients, the 6th-hour inflammatory migration were diminished greatly (Fig. 3), amounting to only 4% of control numbers. The few responding leukocytes were neutrophils and monocytes (Fig. 4). The 6th-hour leukocytic exudate under similar circumstances in the two propositi of prekallikrein (PK) or Fletcher factor deficiency showed a mild increase in cellularity (Fig. 5) over that induced by trauma in the Factor XH-deficient subjects. Although there was a fivefold increase in migrating forms (Fig. 5) over those found in the Hageman factor deficient volunteers (Fig. 3), the quantitative outpouring at this time was less than onefifth of the normal amount depicted in Figure 1. Neutrophils and monocytes again comprised the exudative cells (Fig. 6). On the other hand, the propositus of high molecular weight kininogen (HM r K) or Fitzgerald factor deficiency responded in a surprisingly exuberant and abnormal fashion with excessive numbers of exudative cells (Fig. 7), four times that found in normal controls, more than one hundred times that found in Factor XII deficiency, and more than twenty-fold that found in PK deficiency. The cellular migrations differed quantitatively from the above studies in that, in addition to the neutrophils and monocytes described above, eosinophils and occasional basophils and lymphocytes appeared (Fig. 8). Upon employment of antigenic stimulation at the test site, control lesions were marked at both six and 24 hours by increased cellular migrations in comparison to those brought on by the trauma of the technic alone. Indeed, after three hours, the cellularity covered the entire test site. As previously described, the cells comprising the exudate at 24 hours consisted predominantly of large mononuclears in the form of activated monocytes and transformed lymphocytes (Fig. 9). In contrast, antigenic stimulation in the three Factor XII deficiency patients resulted in no appreciable quantitative or qualitative improvement in their leukocytic outpourings. Figure 10, the 24th hour of inflammation in the youngest child in this family, depicts the exudative fault common to all affected members, a scant response of neutrophils with an occasional monocyte. Antigenic stimulation of the two propositi of PK deficiency resulted in early diminution of the leukocytic migrations to be replaced at 24 hours (Fig. 11) by a fully cellular exudate that covered the lesion site completely and was composed of the aforementioned activated monocytes and transformed lymphocytes, comparable to those found in controls. Antigenic stimulation of the propositus HM r K deficiency achieved a fully covering exudate by the sixth hour as in normal subjects. The nature of the responding cells was striking because of the presence of large numbers of basophilic granulocytes in company with numerous neutrophils and an admixture of eosinophils and large mononuclears as well (Fig. 12). This strange response replaced a predominantly eosinophilic migration that had developed as early as six hours (Fig. 13) in this patient. Because of the unusual and overwhelming quantitative response achieved as early as six hours with only the trauma of the technic (Fig. 3) and the basophilic granulocytic migration at 24 hours after antigenic stimulation, the test was repeated with similar results. The antigenically stimulated test lesion was followed until the 49th hour (Fig. 14), when the basophilic granulocytes had further increased in number and were accompanied by an influx of lymphocytes. Discussion From the foregoing we have seen greatly diminished leukocytic responses in the Factor XH-deficient patients; moderate depression of the early inflammatory leukocytic migrations in PK deficiency, followed by a delayed but eventual restoration to normalcy at 24 hours; while in sharp contrast, excessive leukocytic outpourings pertained in HM r K deficiency, accompanied by qualitatively abnormal aberrations, the latter particularly after antigenic stimulation. Since procoagulant activity leading to fibrin formation is not only the goal of the intrinsic clotting mechanism but also serves to wall off the infectious agents in the inflammatory area as a defensive mechanism in the tissues, it is also important that such an enclosing fibrin wall be eventually dissolved or replaced, once the reparative process is invoked after successful destruction of the invading microorganisms by the leukocytic defenses. Activation of plasminogen (profibrinolysin) to plasmin (fibrinolysin) as dependent in turn on activation of Factor XII, was recognized as early as and amply confirmed. 18 Whether the actual activation is accomplished by activated Factor XII itself or by activated Factor XII in turn activating PK to kallikrein, which latter accomplishes the actual change of plasminogen to plasmin, has been the subject of controversy. There now appears to be convincing evidence at least in vitro, that PK is a Factor XH-dependent plasminogen proactiva-

6 REBUCK 410 % ff{ A.J.C.P. April 1983

7 Vol. 79 No. 4 H. P. SMITH AWARD LECTURE 411 FlG. 9 (upper, left). Twenty-four hours of inflammation in a normal control after antigenic stimulation (0.05 ml diphtheria-tetanus toxoid added to site at 0 hr). The leukocytic sequence after antigen at this stage consists predominantly of large mononuclears (transformed lymphocytes and activated monocytes). Leishman's stain (X 1.000). Fie 10 {upper, right). Patient with Factor XII deficiency. Twenty-four hours of inflammation after antigenic stimulation as in Figure 9. This high magnification shows failure of the normal large mononuclear sequence to develop; the few responding cells are neutrophils and an occasional monocyte. The cellularity in the antigenically stimulated normal (not shown) covered the entire test site, unlike the response to trauma alone; in the Hagenian factor deficient patient shown here, the cellularity of low power (not shown) remained greatly diminished as in Figure 3. Leishman's stain (XI,000). FIG. 11 (lower, left). Propositus of PK deficiency. Twenty-four hours of inflammation after antigenic stimulation as in Figure 9. Cellularity at low power (not shown) surprisingly was restored to normal and covered the entire lesion area and equalled the normal response. Similarly this higher magnification depicts the normal predominant large mononuclears. Leishman's stain (X 1,000). FIG. 12 (lower, right). Propositus of HMrK deficiency. Twenty-four hours of inflammation after antigenic stimulation, same as in Figure 9. Cellularity quantitatively was full and normal in numbers covering the entire lesion, but this high magnification reveals basophilic granulocytes almost as numerous as neutrophils, with an admixture of eosinophils and large mononuclears. Leishman's stain (X 1,000). tor.19 In vivo, failure of activated Factor XII in Factor XH-deficient patients to activate PK would lead in any event to impaired generation of plasmin, which subsequently activates the complement cascade, 15 ' 822 with its several chemotactically important fragmentation products, and similarly to impaired fibrinolysis with its che- motactically active peptides.30 Finally with diminished activation of PK to kallikrein, in the Factor XH-deficient patient, the important chemotactically active product in this interaction, 14 ' 24,4345 kallikrein, is impaired in its generation. Our direct observations in vivo confirm the preliminary report of Gallin and Kaplan (quoted by FlG. 13 (left). Propositus of HMrK deficiency, same lesion as Figure 12 but at 6 hours of inflammation. Eosinophils exceed neutrophils. Leishman's stain (XI,000). FlG. 14 (right). Propositus of HM,K deficiency, same lesion as Figure 12 but at 49 hours of inflammation. Basophilic granulocytes are predominant amid lymphocytes and a few neutrophils. Leishman's stain (XI.000).

8 412 REBUCK. A.J.C.P. April 1983 Gallin and Wolff 8 ) that Factor XH-deficient plasma exhibited impaired chemotactic activity. There is little wonder, then, that Factor XH-deficient patients exhibited such a paucity of inflammatory exudative cells (Figs. 3, 4, and 10). The moderate depression of leukocytic migrations in the PK-deficient patient, which was observed at six hours (Figs. 5 and 6), was in agreement with the in vitro findings of Kaplan and his colleagues 13,42 that such deficiency resulted in decreased chemotaxis. The later restoration of quantitative and qualitative responses to normalcy at 24 hours of inflammation (Fig. 11) reconciles the report of Hathaway and his associates' 2 that skin window tests at 24 hours in their PK-deficient patients did not differ from their normal controls. In this connection, it should be remembered that restoration of procoagulant and fibrinolytic deficits of PK deficient patients' plasma occurs after prolonged incubation, although kinin generation remains impaired. Recently Poon and his colleagues 23 studied chemotaxis in vivo by our method in a patient with PK deficiency with additional partial deficiency of Factor XII. The patient had a history of frequent epistaxis. In vivo chemotaxis of leukocytes with trauma alone was defective and similar to that we have described for the PK propositi above, but in vitro chemotaxis in response to the patient's own serum activated with zymosan and in the skin window to which antigen was applied was normal. One explanation of the paradoxically exuberant leukocytic response we observed in HM r K (Fitzgerald factor) deficiency (Fig. 7) is that Factor XII and PK-kallikrein pathways, although functionally impaired by the lack of complexing HM r K, 89l7-32 are still able to activate plasmin-complement chemotaxis, 33 ' 38 " 41 fibrin-product chemotaxis, 3040 and kallikrein chemotaxis. 13 ' 23 ' 42 ' 45 Such a finding (Fig. 7) suggests that HM r K contributed an important physiologic sign-off signal or mechanism, which concurrently stopped further leukocytic migrations as well as stopping contact activation of procoagulant factors. Lacking such a sign-off, the exuberance of chemotactic activity found in HM r K deficiency is explained. Eventual cessation of leukocytic outpouring and procoagulant activation does occur. The most likely alternate pathways for such eventual sign-off would be from liberation of cellular enzymes in vivo at the site of tissue injury, which in each of the contact activation factor deficiencies, could substitute for the individual missing factor by generation of the required serine protease from the next zymogen in the sequence. Recently Scicli and his colleagues 32 were able to demonstrate that the histidine-rich fragments with highly positive charges, resulting indirectly after the cleavage of kinin from the linear HM r K molecule by kallikrein, actually inhibited the clotting time of normal human plasma. It is appropriate to speculate that the same fragments released from the breakdown of HM r K, independent of the terminal events in the contact activation system, negate the negative charge of injury at the inflammatory site and, thus, preclude any further leukocytic chemotaxis. Using an entirely different biophysical approach to the same problem, Vroman and his fellow workers 35 produced evidence that the first event leading to platelet adherence was adsorption of fibrinogen to which the platelets adhere in turn preferentially. However, HM r K replaced the initially adsorbed fibrinogen on the wettable surface during surface activation of clotting, and platelets, failing to find fibrinogen, would not adhere. Their additional finding that HM r K-deficient plasma failed to replace the originally adherent fibrinogen would permit abnormally prolonged leukocytic margination and diapedesis (Fig. 7) as well as the prolonged state of platelet adherence. The plasminogen-bearing role of eosinophils as demonstrated by Riddle and Barnhart 31 could explain the marked outpouring of eosinophils found in the HM r K-deficient patient (Fig. 13 at 6 hr.). There is a suspicion that a deficiency imposed upon the fibrinolytic mechanism brought about by the HM r K deficiency in some way induced excessive migration of such plasminogen-carrying cells. Upon failure of the eosinophilic flood to compensate for such a loss, the heparin-bearing basophilic granulocytes 1527 (Figs. 12 and 14) seem to have been thrown into the anticoagulant gap occasioned by the loss of the histidine-rich anticoagulant breakdown products 32 of the deficient HM r K. An additional invocation of the law of mass action may be seen in viewing the massive basophilic granulocyte migration (Figs. 12 and 14) as supplying an excess of kallikrein activity, since basophils have been reported as showing kallikrein-like function. 21 Perhaps it is merely the leukocytic proteases of the different lysosomal constituents of the neutrophilic, eosinophilic, and basophilic granulocytes that are invoked, in turn, in the compensatory attempt. I7-20 ' 27 ' As with most chemical messengers of the inflammatory mediation process, we are left with incomplete answers, but it appears from the data we have supplied herein that the contact activation system with Factor XII (Hagefnan factor) as the key protein, 44 is a powerful mediatory of the inflammatory process activating PK (Fletcher factor) and HM r K (Fitzgerald factor) and fibrinolysis with the resultant complement activation, coagulation, chemotaxis, and sign-off, so essential to the inflammatory defenses. Furthermore, patients deficient in each of the proteins of the contact activation system respond with quantitatively manipulated and qualitatively deviated inflammatory cellular exudates, each according to the role played by the missing member. References 1. Benditt EP: Participation of basophils in inflammation. Blood 1961; 18: Bianco C, Eden A, Cohn ZA: The induction of macrophage

9 Vol. 79 No. 4 H. P. SMITH AWARD LECTURE 413 spreading. Role of coagulation factors and the complement system. J Exp Med 1976; 144: Cochrane CG, Griffin JH: The biochemistry and pathophysiology of the contact system of plasma. Advances in Immunology. Edited by H Kunkel, FJ Dixon. New York, Academic Press, 1982, Colman RW, Bagdasarian A, Talamo A, et al: Human kininogen deficiency with diminished levels of plasminogen proactivator and prekallikrein associated with abnormalities of the Hageman factor-dependent pathways. J Clin Invest 1975; 56: Colvin RB, Dvorak H: Role of the clotting system in cell mediated hypersensitivity. J Immunol 1975; 114: Donoldson VH, Glueck HI, Miller MA, Movat HZ, Hamal F: Kininogen deficiency in Fitzgerald train: Role of high molecular weight kinogen in clotting andfibrinolysis.lab Clin Med 1976; 87: Gallin JL, Wolff SM: Leukocyte chemotaxis: physiological consideration and abnormalities. Clin Haematol 1975; 4: Donaldson VH, Kleniewski J, Saito H, Sayed JK: Prekallikrein deficiency in a kindred with kininogen deficiency and fitzgerald trait clotting defect. Evidence that high molecular weight kininogen and prekallikrein exist as a complex in normal human plasma. J Clin Invest 1977; 60: Griffin JH, Cochrane CG: Mechanisms for the involvement of high molecular weight kininogen in surface-dependent reaction of Hageman factor. Proc Natl Acad Sci (USA) 1976; 73: Graham RC, Ebert RH, RatnoffOD, Moses JM: Pathogenesis of inflammation, II. In vivo observations of the inflammatory effects of activated Hageman factor and bradykinin. J Exp Med 1965; 121: Hathaway WE, Belhasen LP, Hathaway HS: Evidence of a new plasma thromboplastin factor. I. Case report, coagulation studies and physicochemical studies. Blood 1965; 26: Hathaway WE, Wuepper KD: Clinical and physiologic studies of two siblings with prekallikrein (Fletcher factor) deficiency. 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