Purified precursor Hageman factor has been demonstrated to bind to soluble bacterial lipopolysaccharide (LPS, endotoxin) isolated from Escherichia coli 0111:B4, and this complex has been shown to have the capacity to convert prekallikrein to its active form. In addition, LPS-activated Hageman factor substantially reduces clotting times in XII-deficient plasma. The capacity to activate Hageman factor has been demonstrated to reside in the lipid A region of the LPS molecule. Activation of Hageman factor by LPS contrasts with fluid-phase activation (e.g., by kallikrein or trypsin) in that no cleavage to lower molecular weight fragments occurs. High concentrations of LPS inhibit the activity of Hageman factor, probably by a direct LPS-Hageman factor interaction.

We have studied the leukocyte-dependent mechanism of histamine release (LDHR) from rabbit platelets, a complement-independent mechanism which has been implicated in the deposition of immune complexes in acute serum sickness of rabbits. It was found by chromatography and passive transfer of serum from immunized rabbits that the antibody responsible for the LDHR was of IgE type. By electron microscope study of the reaction, the leukocyte involved in agglutination of platelets and release of their histamine content was identified as the basophil. Upon addition of antigen, basophils sensitized with IgE degranulated, released their histamine content and a platelet-activating factor (PAF) that caused aggregation of platelets and release of their histamine. Conditions of preparing and preserving PAF activity and some properties of this factor have been elucidated. LDHR must, therefore, be considered as an immediate hypersensitivity-type mechanism which may link allergic reactions with immunologic disease associated with severe structural injury.

The precursor of the kinin-forming enzyme, prekallikrein, was isolated from rabbit plasma protected from activation during preparatory procedures. Prekallikrein was shown to be a 4.5S γ 1 -glycoprotein with an isoelectric point of 5.9 and a mol wt of 99,900. The proenzyme was activated at neutral pH by an enzyme from rabbit or human plasma we have termed prekallikrein activator (PKA) or by trypsin. Prekallikrein was activated by PKA by a process of enzymatic scission. This resulted in the appearance of two fragments; the larger of these possessed kallikrein activity.

The mechanisms reponsible for the deposition of circulating immune complexes have been analyzed. An active process appears to be responsible in both a laboratory model in guinea pigs and in acute immune complex disease (serum sickness) in rabbits. In rabbits, after the injection of antigen to induce serum sickness, immune complexes appear in the circulation. In addition, homocytotropic (IgE) antibody is formed which binds to the surface of basophils. Leukocyte suspensions containing these basophils, when combined with specific antigen, release a soluble factor that causes clumping of platelets and release of their vasoactive amines. An excellent correlation was found between the presence of this mechanism of release of vasoactive amine and the deposition of immune complexes in serum sickness of rabbits. Antagonists of vasoactive amines or depletion of platelets, the major circulating reservoir of these amines, suppressed the deposition of circulating immune complexes and inhibited glomerulitis and arteritis. Upon entering the walls of vessels, the complexes became lodged immune complexes, greater than 19S in size, were deposited along the membranes. The data suggest that at a time when immune complexes appear in the circulation of an immunized rabbit, vasoactive amines are released from platelets in areas where turbulence of blood occurs. Sensitized basophils participate in the release of vasoactive amines from the platelets. The amines induced increased vascular permeability which leads to deposition of large complexes from the circulation in vessel walls by a process of filtration. The deposited complexes then induce inflammatory injury.

A small fragment of C3, called C3a, which has smooth muscle contracting activity, was isolated by three different methods. At pH 8.6, C3a behaved as cation, and using the Archibald method, its mol wt was determined to be 7000. A specific antiserum to C3a showed the fragment to be antigenically distinct from the rest of the C3 molecule, i.e., the C3b portion. The same antiserum and an anti-whole C3 were able to inhibit the biologic activity of C3a. In addition to anaphylatoxin activity, leukocyte chemotactic activity was shown to reside in C3a. Treatment with trypsin caused the cationic fragment to become anionic and abolished the anaphylatoxin but not the chemotactic activity. C3a fragments with identical biologic activity and comparable cationic properties, as determined by acid disc electrophoresis, were obtained by treatment of C3 with C3 convertase, C3 inactivator complex, trypsin, and plasmin. Thrombin produced a similar C3 fragment which was inactive. It was concluded that C3a corresponds to an unusually basic portion of C3 which may be liberated by attack of a variety of enzymes on a highly susceptible region of the native C3 molecule. C3b was cleaved by trypsin and less efficiently by thrombin or plasmin into two antigenically distinct pieces: the larger C3c fragment corresponding to ß 1A and the smaller C3d fragment to α 2D of aged serum. The c- and the d-fragments were separated and characterized. Isolated C3a rapidly lost its anaphylatoxin activity when treated with small amounts of a partially purified, thermolabile 10S α-pseudoglobulin of human serum. The conditions of inactivation suggested an enzymatic reaction. The anaphylatoxin inactivator also destroyed the activity of C5-derived anaphylatoxin and of lysyl bradykinin.

The relationship between certain physicochemical properties of circulating immune complexes and their ability to localize in vessel walls during a state of increased permeability was studied. The ability to become deposited was related to the large size of complexes, rather than to their net charge or to a specific affinity between complexes and structures of vessel walls. Soluble complexes with sedimentation rates greater than 19S were capable of being entrapped along the vessel wall membranes, while complexes smaller than this were not. These large complexes were removed rapidly from the circulation, while smaller complexes persisted. Minimal levels of total complexes in the circulation necessary for detectable vascular localization were found to be as low as 15 µg antibody N /ml plasma. In experimental serum sickness, a disease known to be induced by circulating immune complexes, the development of vascular and glomerular lesions occurred almost exclusively in rabbits having large (greater than 19S) circulating immune complexes. Animals with smaller complexes did not show deposition of complexes in glomeruli or development of glomerulonephritis. Their incidence of vasculitis was markedly reduced.

In serum sickness, mechanisms by which circulating immune complexes become localized in the walls of vessels and glomeruli have been studied. In affected arteries, morphologic observations showed that circulating marker particles of carbon would rapidly deposit along the luminal surface of the internal elastic lamina. This, as in previous studies, suggested an increase in vascular permeability during which large molecules were capable of being trapped by a filtering membrane in the vessel wall. In attempts to prevent the increase in vascular permeability, rabbits were treated with antagonists of histamine and serotonin. Such treatment markedly inhibited the localization of immune complexes in glomeruli, the development of proteinuria, and glomerular endothelial proliferation. Cardiovascular lesions also were largely prevented from developing. Depletion of platelets, the principal reservoir of vasoactive amines, had a similar though less pronounced effect. While the deposition of immune complexes was inhibited, allergic inflammation in general was not, since normal rabbits treated as above were found capable of developing full Arthus reactions and acute nephrotoxic nephritis. Hydrodynamic factors were noted to be important in determining the location of arterial lesions. Studies of aortas from unmodified rabbits and from those with surgically induced coarctations of the abdominal aorta revealed intimal lesions concentrated at areas of high turbulence, such as at branches, bifurcations, outflows and zones of configurational change. Lesions in these areas were also largely inhibitable by depletion of platelets or by antagonists of histamine and serotonin.

Vascular basement membrane was disrupted in the presence of polymorphonuclear leukocytes (PMN's) during two immunologic reactions: The Arthus phenomenon and the reaction to locally injected antibody to vascular basement membrane. This disruption was evidenced by ( a ) the inability of the basement membrane to retain circulating carbon, by ( b ) loss of antigenic constituents, and by ( c ) electron microscopic observation showing actual gaps in the structure of the vascular basement membrane. The factors within PMN's responsible for damage to isolated glomerular basement membrane in vitro were found by isolation procedures to be cathepsins D and E. Cationic proteins of PMN's were separable from the cathepsins. While inducing vascular permeability upon injection, these basic proteins failed to inflict the severe damage to the basement membrane observed in Arthus and antibasement membrane reactions. It is concluded that the full expression of these immunologic lesions requires destruction of the basement membrane possibly brought about by cathepsins D and E. Some of the physicochemical properties of these pathologically active leukocytic factors are given.

By the use of chambers containing two compartments with an interposed micropore filter, chemotaxis of polymorphonuclear leukocytes (PMN's) in vitro was studied employing various agents that fixed serum complement (C'). Antigen-antibody complexes, zymosan, and aggregated human gamma globulin, in the presence of fresh rabbit, guinea pig, or mouse serum resulted in the migration of PMN's through the micropore filter. Pepsin-degraded rabbit antibody or unaltered duck serum containing antibody did not exhibit such activity after addition of antigen. Heating of the serum before treatment or the presence of EDTA prevented the generation of the chemotactic factor. The chemotactic factor could not be generated in whole serum from rabbits genetically deficient in C'. However, the defect in this rabbit serum could be corrected by addition of rabbit or human C'6. Serum of B10·D2 mice deficient in hemolytic C' also yielded poor chemotactic activity. Interaction of the first four reacting components of guinea pig C' did not result in significant chemotactic activity unless guinea pig euglobulin with heat labile components was also present. In rabbit serum, C'5 and C'6, when "activated" by interaction with the first four reacting components, behaved like a protein-protein complex and exhibited marked chemotactic activity. By employing conditions favoring dissociation of the complex, the individual components were isolated and shown to be chemotactically inactive. Upon recombination of the two components, however, activity reappeared. Using another approach, the C'5–C'6 complex was isolated intact, and shown to be chemotactically active while other fractions not containing these components were not active. It is postulated that the C'5–C'6 complex is the active chemotactic factor generated in serum after the addition of C'-fixing agents.

The present studies suggest that polymorphonuclear leukocytes (PMN's) are essential for the development of cardiovascular lesions in serum sickness. In the absence of PMN's, necrotic vascular lesions were never seen and endothelial proliferation in arteries was inhibited. Zones of fibrinoid deposits did not occur. By contrast, at least two-thirds of the control animals exhibited endothelial proliferation, and half had necrosis of arterial walls, usually with fibrinoid deposits. In arterial lesions that involved the intima and media, the internal elastic lamina was disrupted. This was associated with accumulations of PMN's and was prevented when PMN's were depleted. The observations suggested that the elastic lamina acts as a barrier to the outward spread of inflammation in arteries and that it is an important substrate of PMN action. Although glomerulitis and proteinuria developed in PMN-depleted animals, no conclusions could be drawn concerning the pathogenic role of PMN's in renal lesions, since PMN depletion could not be effected before the onset of immune elimination without influencing the immune response itself. Host complement (ß1C-globulin) was localized along with the antigen and rabbit gamma globulin in glomeruli and arteries showing lesions. In the glomeruli these deposits formed a granular lining along the area of the basement membrane. In arteries the fluorescent amorphous particles were in the intima and media of inflamed vessels. The immune response to BSA and the incidence and severity of cardiovascular and renal lesions were enhanced by the intravenous administration of pooled rabbit antiserum to BSA given 18 hours before BSA antigen and by injecting endotoxin along with the BSA. These additions to the usual procedure of inducing serum sickness did not appear to change the quality of the disease. In normal rabbits, the peak incidence of cardiovascular lesions was early in immune elimination of antigen, at a time when levels of circulating complexes was maximal. Conversely, the severest renal injury was noted several days later, at the completion of immune elimination.

In acute nephrotoxic nephritis, polymorphonuclear leukocytes (polymorphs) accumulated in large numbers in the glomeruli in the first 12 hours. The endothelial cells were dislodged by the polymorphs which then came to lie immediately adjacent to the glomerular basement membranes. Ultrastructural changes in neither polymorphs nor basement membranes were observed. Depletion of polymorphs in both rats and rabbits prevented the development of proteinuria. This occurred when doses of nephrotoxic globulin were employed that produced proteinurias of as much as 1800 mg/kg/24 hours in intact rabbits, or enough to yield near maximal immediate proteinuria in intact rats. In addition, measurable glomerular damage was frequently averted until the onset of the secondary stage of NTN. Controls indicated that the polymorph depleted animals exhibited minimal non-specific changes in the blood, that the ability of their vascular beds to react to stimuli was not affected, and that deposition of nephrotoxic antibody and C' in the glomeruli was not inhibited. Elimination of polymorphs from the circulation was only partially effective in preventing glomerular damage when large doses of nephrotoxic globulin were used. This indicated that under these circumstances, a polymorph independent glomerular injury may also take place in first stage nephrotoxic nephritis. An indirect role of C', i.e ., the accumulation of polymorphs, in bringing about glomerular injury in first stage nephrotoxic nephritis was apparent. When rabbit nephrotoxic globulin was injected into rats depleted of C', or when duck nephrotoxic globulin that fixed C' poorly was injected into normal rats, C' failed to bind with the antibody along glomerular basement membranes and polymorphs did not accumulate.

Rats and guinea pigs were depleted of complement (C') by treatment with heat aggregated human γ-globulin (agg HGG), zymosan, anti-ß1C globulin, and carrageenan. Although antigen and antibody were bound to vascular structures, Arthus reactions were inhibited. This inhibition was characterized by the lack of C' binding to walls of vessels, the lack of polymorphonuclear (PMN's) cellular infiltrates, and the lack of significant vascular damage. When the same animals were followed for several hours thereafter, levels of serum C' began to rise, C' was bound in tissues, PMN infiltrates appeared, and immunologic vasculitis developed. Blood counts, chemotaxis of PMN's induced by lysates of PMN granules, together with studies on motility and phagocytosis by PMN's obtained from C' depleted rats, failed to establish any abnormality in these cells which would account for inhibition of Arthus reactions. The specificity of C' depletion in terms of effects in the first four reacting components of guinea pig C' was studied. Treatment with agg HGG led to loss of activity in all components, whereas zymosan and anti-ß1C globulin predominately affected the third component (C'3c). Carrageenan mainly affected the first two reacting components of C'. Thus, the availability of the 3c component, or a subsequently reacting component, correlated with the attraction of PMN's to immune reactants in vivo . Various antibodies with different C' fixing capacities in vitro were tested for their ability to induce immunologic vasculitis in normal animals. In rats, only those antibodies which fixed C' in vitro possessed biological activity, whereas in guinea pigs, all antibodies tested, regardless of C' fixation in vitro , induced Arthus reactions. For a given antibody in rats the vasculitis-inducing property was reflected in its ability to bind C' in vascular structures. Rats depleted of circulating PMN's by specific antibody were tested for Arthus activity. Although concentrations of immune reactants and C' were readily detected in vascular structures, no PMN infiltration occurred and significant vascular damage was averted.

Localization of circulating antigen-antibody complexes in vessels of guinea pigs by means of anaphylactic shock was found to be mediated by histamine that was released at the time of anaphylaxis. The source of the histamine may have been the mast cell as noted in studies employing a direct attack on the mast cells by octylamine. Platelets played apparently little to no role in guinea pigs in the anaphylactic deposition of circulating complexes. Rat anaphylatoxin was found to cause vascular localization and symptoms of anaphylaxis identical with that brought about by antigen-antibody anaphylaxis. This also was found to be dependent upon the release of histamine. Antibody against Forssman antigen in the vessel walls of the guinea pigs also led to deposition of circulating complexes. This was found not to be histamine dependent. The possible role of local increase in vascular permeability in certain experimental disease states in the localization of circulating complexes is discussed.

A short term model in which circulating antigen-antibody complexes and host complement localized in vessel walls of guinea pigs was analyzed. Localization was accomplished by subjecting the animals to anaphylactic shock. The circulating macromolecules, such as antigen-antibody complexes, appeared to localize by being trapped in the vessel wall along the basement membrane that acted as a filter during a state of increased permeability of the vessel. It was suggested that this point of localization between the endothelial cell and the basement membrane may well represent the earliest focus of inflammation in diseases caused by the deposition of injurious macromolecules such as soluble antigen-antibody complexes from the blood stream. Complexes localized in the vessel walls did not provoke Arthus-type vasculonecrotic reactions even though in both these vessels and in cutaneous Arthus reactions antibody, antigen, and host complement (C'3c) were deposited in the vessel walls. The possibility was presented that since circulating macromolecules and probably complexes deposited in ( a ) relatively small amounts, and ( b ) in a position beneath endothelial cells, they were not strongly chemotactic toward circulating polymorphonuclear leukocytes. Vasculonecrotic reactions, therefore, were not observed. It was brought out that this may be similar to the situation in glomerulonephritis induced by localized immune complexes, in which severe necrosis is not observed. In the Arthus vascular reaction, host complement was found microscopically accumulated with the immune reactants in affected vessel walls.

The role of polymorphs in the Arthus type hypersensitivity vasculitis has been studied. Polymorphs were found to play an essential role in not only producing the inflammatory vasculitis, but also were instrumental in ridding the damaged vessel of the antigen, probably by means of proteolytic catabolism at the inflammatory site. A temporal relationship between the disappearance of antigen from the damaged vessels and a decrease in inflammatory reaction was found. The earliest localization of antigen and its associated rabbit globulin in the Arthus vasculitis was found to be beneath the endothelium of small vessels. Since submitting this article for publication, Sorkin and Boyden ( J. Immunol ., 1959, 82 , 332) have reported the catabolism of antigen in the presence of antibody by mononuclear cells obtained from the peritoneal cavities of guinea pigs. Evidence was presented indicating that the antigen molecule was actually broken down by the mononuclear cells. Techniques similar to those reported here were used.

The in vivo activity of soluble antigen-antibody complexes was tested by a single intradermal injection in rabbits. Skin reactions were obtained marked by erythema, induration, and occasionally hemorrhage and necrosis. Microscopically, diffuse inflammation and occasional vascular necrosis could be found at all dosages. This indicates that soluble antigen-antibody complexes are phlogogenic and provides support for the suggestion that complexes are responsible for the lesions seen in serum sickness. The reactions were similar in severity to local passive Arthus (LPA) reactions at equal dosages of antibody in the dosage range studied. BSA antigen could be found in large concentrations in affected vessel walls of both reverse passive Arthus (RPA) and active or classical Arthus reactions. It is suggested that this predominantly vascular localization of antigen might bring about the relative severity of the RPA and active Arthus reactions, as contrasted to the complex and LPA reactions. The finding of affected vessels in the complex and LPA reactions containing little or no antigen and antibody, while these components were present in adjacent areas, suggests that the antigen-antibody combination may cause vascular reaction and damage by the release of physiologically active mediators from the tissue or tissue fluid.