Stephen I. Rosenfeld

C5 chemotactic fragment induces leukocyte production of tissue factor activity: a link between complement and coagulation.

Complement-activated human plasma causes generation of tissue factor in human leukocytes. This phenomenon appears to be related to the fifth component of complement (C5) as demonstrated by the use of C5 deficient-plasma and suppression of activity with antibody to C5. Isolation of the chemotactic factor from activated serum or trypsinization of purified C5 reproduces the phenomenon. These data provide evidence for a direct link between complement products and activation of the coagulation system. Because chemotactic peptides from C5 can be generated by a variety of enzymes, our findings suggest a relationship between complement, coagulation, and inflammation.

Hereditary deficiency of the fifth component of complement in man. I. Clinical, immunochemical, and family studies.

The first recognized human kindred with hereditary deficiency of the fifth component of complement (C5) is described. The proband, a 20-year-old black female with systemic lupus erythematosus since age 11, lacked serum hemolytic complement activity, even during remission. C5 was undetectable in her serum by both immunodiffusion and hemolytic assays. Other complement components were normal during remission of lupus, but C1, C4, C2, and C3 levels fell during exacerbations. A younger half-sister, who had no underlying disease, was also found to lack immunochemically detectable C5. By hemolytic assay, she exhibited 1-2% of the normal serum C5 level and normal concentrations of other complement components. C5 levels of other family members were either normal or approximately half-normal, consistent with autosomal codominant inheritance of the gene determining C5 deficiency. Normal hemolytic titers were restored to both homozygous C5-deficient (C5D) sera by addition of highly purified human C5. In specific C5 titrations, however, it was noted that when limited amounts of C5 were assayed in the presence of low dilutions of either C5D serum, curving rather than linear dose-response plots were consistently obtained, suggesting some inhibitory effect. Further studies suggested that low dilutions of C5D serum contain a factor (or factors) interfering at some step in the hemolytic assay of C5, rather than a true C5 inhibitor or inactivator. Of clinical interest are (a) the documentation of membranous glomerulonephritis, vasculitis, and arthritis in an individual lacking C5 (and its biologic functions), and (b) a remarkable propensity to bacterial infections in the proband, even during periods of low-dose or alternate-day corticosteroid therapy. Other observations indicate that the C5D state is compatible with normal coagulation function and the capacity to mount a neutrophilic leukocytosis during pyogenic infection.

Penicillin resensitization among hospitalized patients

The purpose of this study was to determine the frequency of resensitization to penicillin after oral or intravenous treatment with beta-lactam antibiotics in hospitalized patients with histories of penicillin allergy. Seventeen adults (aged 24 to 76 years) and one child (aged 10 years) were treated intravenously and/or orally with beta-lactam antibiotics after negative skin tests were obtained with benzylpenicilloyl polylysine, potassium penicillin G, and alkaline hydrolysis products of penicillin G as minor determinant mixture. Repeat skin testing was performed 1 to 12 months after the therapy. Three patients (16%) became skin test positive after the treatment. Two patients reacted to potassium penicillin G alone, and the other patient reacted to benzylpenicilloyl polylysine and minor determinant mixture. These three patients were among the 15 patients who were treated with intravenous antibiotics. This study reveals a high percentage of skin test conversion after intravenously administered penicillin therapy and confirms the present practice of advising patients with a history of penicillin allergy who have successfully completed penicillin treatment to have a repeat skin test before future exposure to beta-lactam antibiotics.

Structural polymorphism of the fourth component of human complement

The fourth component of human complement (C4) in 102 individual plasma samples has been examined by the technique of antigen-antibody crossed electrophoresis (AACE). Electrophoretic heterogeneity of C4 was manifested by the repeated occurrence of seven different precipitin patterns. These patterns were formed by varying combinations of three subtypes of C4, differing in electrophoretic mobility. The subtypes were designated C, A, and A(1), in order of increasing electrophoretic mobility toward the anode. The evidence that the observed electrophoretic heterogeneity of the C4 molecule represents structural polymorphism rests on five points: the pattern obtained from the plasma of a given individual was reproducible in different runs and with different bleedings; all seven patterns could be demonstrated on the same electrophoretic run; C4 of a given subtype retained its characteristic mobility after purification, when run alone or mixed with plasma containing C4 of other subtypes; the subtypes A(1) and C comprising pattern 6 could be separated chromatographically as well as electrophoretically; and the characteristic relative mobilities of different C4 subtypes, in plasma or after purification, were retained even after the rather large shift in mobility associated with conversion to C4i. The ratio of C4 hemolytic activity to protein concentration varied according to the subtype composition of individual samples, with highest ratios occurring with patterns composed of subtype C alone, intermediate values with patterns consisting of A and C, and lower values occurring with patterns containing subtype A alone. Although the mechanism of inheritance of this polymorphism is not yet clear, the data suggest that subtypes A and A(1) are inherited as autosomal codominant characteristics, independent of the inheritance of subtype C.

Linkage between the gene (or genes) controlling synthesis of the fourth component of complement and the major histocompatibility complex.

In an attempt to map the gene (or genes) controlling the synthesis fo the fourth component of complement (C4), we performed linkage studies in a family with hereditary C4 deficiency. The proband, a seven-year-old boy with lupus erythematosus, consistently lacked deteftable serum C4 by both functional and protein measurements. The complement defect was transmitted as an autosomal recessive disorder. Eight of 15 family members were considered to be heterozygotes, seven because of low C4 levels and one because of genetic data (obligate heterozygote). The gene (or genes) coding for C4 deficiency appeared to be linked to the major histocompatibility complex (A2,B12,DW2 on the maternal side and A2,BW15,LD108 on the paternal side) and to other markers known to be in close proximity to the histocompatibility complex on chromosome 6 (phosphoglucomutase-3, glyoxalase-1 and properdin factor B).

Inhibition of the Lytic Action of Cell-bound Terminal Complement Components by Human High Density Lipoproteins and Apoproteins

Human serum lipoproteins are known to participate in or modify several immunologically relevant responses, including the inhibition of target cell lysis initiated by fluid-phase C5b-7 (reactive lysis). We now report that human high density lipoproteins (HDL) can inhibit the complement (C) lytic mechanism after C5b-7, C5b-8, and even C5b-9 have been bound to the target membrane. This inhibitory activity of serum or plasma copurifies in hydrophobic chromatography with antigenically detected apolipoprotein A-I (apoA-I), the major HDL apoprotein, and with HDL in CsCl density gradient ultracentrifugation. Although HDL is more active than its apoproteins in fluid-phase inhibition of C5b-7-initiated reactive lysis, the HDL apoproteins are more effective after C5b-7, C5b-8, or C5b-9 have become bound to human or sheep erythrocytes (E). Highly purified HDL apoproteins, apoA-I and apoA-II, both have greater inhibitory activity than whole HDL on a protein weight basis, and some evidence has been obtained that apoA-I dissociating spontaneously from HDL may be the principal inhibitory moiety in physiological situations. HDL lipids themselves are inactive. The HDL-related inhibitors are ineffective when incubated with EC5b-7 and removed before C8 and C9 are added, and only minimally effective on cell-bound C5b-8 sites before C9 is added. They exert their most prominent inhibitory activity after C9 has been bound to EC5b-8 at low temperature, but before the final temperature-dependent, Zn(++)-inhibitable membrane damage steps have occurred. Therefore, HDL or its apoproteins do not act to repair already established transmembrane channels, but might interfere either with insertion of C9 into the lipid bilayer or with polymerization of C9 at C5b-8 sites. This heat-stable inhibitory activity can be demonstrated to modify lysis of erythrocytes in whole serum, i.e., it does not depend upon artificial interruption of the complement membrane attack sequence at any of the above-mentioned stages. Contributions of the target membrane itself to the mechanism of inhibition are suggested by the observations that, in contrast to sheep or normal human E, lysis of guinea pig E or human E from patients with paroxysmal nocturnal hemoglobinuria is inhibited poorly. This is the first description of a naturally occurring plasma inhibitor acting on the terminal, membrane-associated events in complement lysis. Although further study is required to assess the physiologic or immunopathologic significance of this new function of HDL, the HDL apoproteins or their relevant fragments should be useful experimentally as molecular probes of the lytic mechanism.

Complement Lysis of Human Erythrocytes: DIFFERING SUSCEPTIBILITY OF TWO TYPES OF PAROXYSMAL NOCTURNAL HEMOGLOBINURIA CELLS TO C5b-9

Although enhanced sensitivity of erythrocytes to complement-mediated lysis is a hallmark of paroxysmal nocturnal hemoglobinuria (PNH), subpopulations of erythrocytes in such patients vary significantly in this respect. One PNH erythrocyte subpopulation (termed type III) comprises exquisitely sensitive cells, whereas type II PNH erythrocytes are intermediate in complement sensitivity between PNH type III and normal human erythrocytes. Differences in the action of the terminal complement components that would account for the differing lytic behavior of types II and III PNH erythrocytes have been proposed but not directly demonstrated. The present studies, making use of carefully selected cases with pure populations of type II or type III erythrocytes, confirm a prior observation that antibody-coated PNH erythrocytes of both types II and III display comparably supranormal C3 binding in whole human serum. However, when lysis was induced by the isolated C5b-9 membrane attack mechanism, bypassing the requirement for C3 binding, only type III PNH cells exhibited greater than normal lysis. This finding suggests that type III PNH erythrocytes have an additional membrane abnormality not present in type II cells. Thus, the differing lytic behavior of these two cell types in whole serum may reflect the additive effects on type III cells of both exaggerated C3 binding and enhanced sensitivity to C5b-9, whereas the more moderate lysis of type II PNH cells may be determined mainly or entirely by the earlier-acting mechanism producing augmented C3 binding. The failure of guinea pig C8 and C9, as opposed to human C8 and C9, to reveal the true lytic sensitivity of PNH-III E in our earlier study is illustrated, and its implications briefly discussed.

Hereditary deficiency of the fifth component of complement in man. II. Biological properties of C5-deficient human serum.

The first known human kindred with hereditary deficiency of the fifth component of complement (C5) was documented in the accompanying report. This study examines several biological properties of C5-deficient (C5D) human serum, particularly sera obtained from two C5D homozygotes. The proband, who has inactive systemic lupus erythematosus is completely lacking C5, while her healthy half-sister has 1-2% of normal levels. Both sera were severely impaired in their ability to generate chemotactic activity for normal human neutrophils upon incubation with aggregated human gamma-globulin or Escherichia coli endotoxin. This function was fully restored in the siblings serum, and substantially improved in the probands serum, by addition of highly purified human C5 to normal serum concentrations. Sera from eight family members who were apparently heterozygous for C5 deficiency gave normal chemotactic scores. The ability of C5D serum to opsonize Saccharomyces cerevisiae (bakers yeast) or Candida albicans for ingestion by normal neutrophils was completely normal. In addition, C5D serum was capable of promoting normal phagocytosis and intracellular killing of Staphylococcus aureus. The probands serum was incapable of mediating lysis of erythrocytes from a patient with paroxysmal nocturnal hemoglobinuria in both the sucrose hemolysia and acid hemolysis tests, and also lacked bactericidal activity against sensitized or unsensitized Salmonella typhi. The siblings serum, containing only 1-2% of normal C5, effectively lysed S. typhi, but only at eightfold lower serum dilutions as compared to normals. These findings underscore the critical role of C5 in the generation of chemotactic activity and in cytolytic reactions, as opposed to a nonobligatory or minimal role in opsonization, at least for the organisms under study.

Recurrent abdominal pain as the sole manifestation of hereditary angioedema in multiple family members

This paper describes a previously unreported finding of abdominal pain as the only lifelong manifestation of hereditary angioedema in multiple family members. This diagnosis was obscured by the absence of cutaneous, oropharyngeal, and respiratory involvement. Barium studies performed during painful attacks showed transient intestinal wall edema which, along with abnormalities in the C4 level and C1 esterase inhibitor activity, confirmed the diagnosis. It is important that hereditary angioedema be recognized in its various forms so that invasive procedures can be avoided and prophylactic therapy can be administered.

beta-adrenergic receptors of human leukocytes: studies with intact mononuclear and polymorphonuclear cells and membranes comparing two radioligands in the presence and absence of chloroquine

Owing to the large differences in reported values for beta-adrenergic receptor numbers and binding affinity in normal leukocytes, we undertook a systematic re-examination of the binding of two widely used beta antagonists, (-)-[3H]dihydroalprenolol (DHA) and (+/-)-[125I]iodohydroxybenzylpindolol (HYP), to intact normal mononuclear (MN) leukocytes and polymorphonuclear (PMN) leukocytes and membrane preparations. Assays were conducted in the presence and absence of chloroquine, which has been proposed recently to eliminate ligand uptake into a non-receptor cell compartment such as lysosomes. The binding curves relating radioligand concentration to specific sitesper intact cell were biphasic. At high (10-24 nM) (-)-DHA ligand concentration in the absence of chloroquine, a large number (20,000-60,000 sites/cell) of low affinity (Kd 12-15 nM) stereospecific binding sites were detected in both cell types. This class of binding sites was eliminated by 10 microM chloroquine not only in PMN cells but also in the lysome-poor MN cells (greater than or equal to 90% lymphocytes), leaving 2000-3000 specific high affinity (-)-DHA sites/cell. In the absence of chloroquine, comparably low numbers of specific high affinity binding sites/cell were also obtained by the use of appropriately low concentrations of (-)-DHA or (+/-)-HYP (800 pM or less). However, even at these low radioligand concentrations chosen to measure high affinity specific binding, the addition of 10 microM chloroquine produced a moderate reduction in the number of sites/cell, without a detectable change in the apparent Kd. Mean (+/- S.E.M.) site numbers obtained in the presence of chloroquine were: 1331 +/- 100 sites/MN cell and 1135 +/- 129 sites/PMN cell (Kd 143-153 pM) using (-)-DHA; and 1487 +/- 210 sites/MN cell and 1065 +/- 69 sites/PMN cell [avg. Kd(+/-) 224-274 pM] using (+/-)-HYP. Chloroquine had no effect on agonist-stimulated cAMP production but produced an apparent increase in the effectiveness of (-)-propranolol as an inhibitor of DHA binding. Competition studies on the binding of DHA and HYP with zinterol and practolol confirmed that the receptor was of the beta 2-subtype for both MN and PMN cells. The detection of a moderately larger number of high affinity binding sites at saturation (Scatchard analysis) by (+/-)-HYP than by (-)-DHA was a consistent finding with either intact cells or membranes, with or without chloroquine. The possible overestimation of receptor numbers by a racemic ligand such as (+/-)-HYP is discussed and leads us to favor the use of a pure stereoisomer such as (-)-DHA. A system employing 800 pM (-)-[3H]DHA, 1 microM (-)-propranolol and 10 microM chloroquine with intact MN and PMN cells yielded reproducible and plausible results. Our values for beta-adrenergic receptor numbers of intact MN and PMN cells and membranes are compared to others in the literature.