James H. Jandl

Red Cells Coated with Immunoglobulin G: Binding and Sphering by Mononuclear Cells in Man

Human monocytes, macrophages, and certain lymphocytes bind firmly to red cells coated with immunoglobulin G, whether or not it is acting as antibody. Monocyte binding is specific for cells coated with immunoglobulin G and is inhibited specifically by this immunoglobulin or its Fc-fragment in solution. Although not involving serum complement and not usually a prelude to erythrophagocytosis, this binding causes rapid morphological injury to red cells, as manifested by their sphering, increased osmotic fragility, deformation, and fragmentation. It is inferred that mononuclear cells have specific surface receptors for immunoglobulin G and that these provide a critical phase of the mechanism in vivo, whereby red cells or other particles coated with antibody are apprehended and destroyed.

Increased Susceptibility to Infection Associated with Abnormalities of Complement-Mediated Functions and of the Third Component of Complement (C3)

Abstract In a patient with Klinefelters syndrome and lifelong increased susceptibility to infection, no abnormalities were found in humoral antibody production, cellular immunity or leukocyte function. In contrast, the patients serum complement-mediated functions were grossly deficient. The concentrations of serum complement components were normal except for that of C3 (β1C-globulin), which was less than one-third normal. The bulk of this was in the form of the inactive conversion product, C3b, at all times that the patients serum or plasma was examined over a two-year period. Addition of small amounts of normal serum, but not purified C3, to the patients serum improved all complement-mediated functions in vitro. This disorder, which may represent an inborn deficiency of a protein necessary for C3 stability in vivo and in vitro, is detected by the lowered serum C3 concentration and a positive non-gamma (C3) Coombs antiglobulin test.

OXIDATIVE HEMOLYSIS AND PRECIPITATION OF HEMOGLOBIN. I. HEINZ BODY ANEMIAS AS AN ACCELERATION OF RED CELL AGING

The propensity of certain chemicals and drugs to induce hemolytic anemias has been a subject of much interest for almost a century (1-4). The majority of substances so active are aromatic compounds containing amino-, nitro-, or hydroxy groups, although a number of inorganic compounds (e.g., hydroxylamines, nitrates, nitrites and chlorates) are also active (1-4). Such compounds usually cause red cell injury without evidence of specific toxicity to other cells or tissues. The hemolytic process is characterized by two rather distinctive features in the affected red cells: 1) the appearance of l)rownish or greenish derivatives of hemoglobin, including methemoglobin, according to most (3) but not all (4) reports; and 2) the formation within red cells of water-insoluble, stainable granules, generally termed Heinz bodies. There has been extensive debate as to the identity and significance of the hemoglobin discoloration following the adiministration of these compounds. Much of the disagreement may be attributed to the fact that many of the observations have been made in vivo, where pigments initially produced by the compound are mixed with the various products of hemoglobin catabolism. Furthermore, the fact that certain of the pigments produced, such as miiethemoglobin, are reversible, whereas certain others (including so-called sulfhemoglobin) are not (3), has undoubtedly led to conflicting observations. In those few studies where early, sequential observations have been made, methemoglobinemia has been striking albeit transient (5, 6). Studies of compounds such as phenylhydrazine, which are active against red cells in vitro as well as in vivo, have shown that hemoglobin may be changed in part to methemo-

THE ANEMIA OF LIVER DISEASE: OBSERVATIONS ON ITS MECHANISM

The frequent association of anemia with chronic liver disease provides a possible means of examining the role of the liver with respect to the formation and destruction of red blood cells. An understanding of the mechanism of this anemia has been retarded by the frequent appearance in afflicted patients of complicating factors such as blood loss, infection, neoplasm, and occasionally nutritional macrocytic anemia, as well as by the diversity of the actual hepatic lesions. Furthermore, the proliferative aspects of the liver and spleen in hepatic cirrhosis render it theoretically likely that in this commonform of chronic liver disease superimposed pathologic, as well as altered physiologic, processes act upon the production, sustenance, and destruction of red cells. Nevertheless, knowledge of the pattern of red cell turnover in chronic liver disease is of basic clinical and physiologic interest. A number of surmises have been offered to account for the pathogenesis of the anemia of liver disease. The hypothesis that this anemia is metabolically similar to pernicious anemia and that it arises from defective storage of the subsequently identified Vitamin Bl2 (1-3) has been generally rejected for several reasons, namely: The morphologic dissimilarities of the two types of anemia (4, 5); the failure of typical cases to respond to preparations containing Vitamin B,2 (6); the demonstration of anti-pernicious anemia activity in the liver of cirrhotic patients with macrocytic anemia (7); and the inability of liver extract to prevent anemia during experimental liver injury (8). More recently it has been suggested that another, unrelated metabolic defect exists (6), possibly exaggerated in some cases

The Agglutination and Scnsitization of Red Cells by Metallic Cations: Interactions between Multivalent Metals and the Red-Cell Membrane *

A PROMINENT fcature of the rcactioii between red cells and most antibodies developed against them is the devclopinent of agglutination or of agglutiiiability which can be subsequcntly elicited under certain conditions. Indeed, agglutination in viva appears to initiate the destruction of red cells by nonhaeniolytic antibodies to red cells in dogs (Castle, Ham and Shen, 1950) and in man (Jandl, 1955; Jandl and Castle, 1956). Over the years, quite apart from antibodies, many substanccs have been observed to agglutinate normal animal or human red cells either by dircct action, as with silicic acid (Landsteiner and JagiE, 1904), tannic acid (Reiner and Fischcr, 1929), various plant agglutinins (Goddard and Mendel, 1929), basic polyaniiiio acids (Rubini, Stahiliami and Rasniusscn, 195 I ) and some viruses or enzymes (Hirst, 1941; Burnct, McCrea and Stonc, 1946; Briody, 1948), or after the subsequent addition of scrum, as following exposure to certain enzymes (Morton and Pickles, 1947), or to periodate (Friedenreich, 1928; Stewart, 1949; Moskowitz and Treffers, 1950). In addition, both silicic acid (Landsteincr and Jagii-, 1904; Landsteiner and Rock, 1912) and tannic acid (Reincr and Fischer, 1929; Peck and Thomas, 1949) have been found to sensitize the red-cell surface to the subsequent addition of complcmcnt, and, in the case of tannic acid, to the addition of properdin (Him and Pillenicr, 1955). Boydeii (1951) and others (Denny aiid Thomas, 1953) observed that iioriiial red cells previously trcatcd with appropriatc concciitrations of taniiic acid possess the property of absorbing many proteins from solutions addcd subsequently to the cell suspension. Such cells were then agglutinable by antisera devclopcd in animals against the adsorbcd proteins. Red cells treated with tetra-azotized bcnzidiiic have bccii fourid to behave similarly (Cole, Matloff and Farrell, 1955), and both tannic acid and tctra-azotized benzidine have been employed cxpcriineiitally for coupling antigens to thc red-ccll surface in order to provide, through the developmcnt of rcd-cell agglutination, a visible ciid-point following the addition of non-precipitating antibodics. The prcparatory effcct of taiinic acid on ccll membranes is also evident in the ability of the cell walls of bactcria trcated with tannic acid to adsorb visibly certain dyes (Ogiiisky and Umbrcit, 1955). Muirhead, Grovcs and Bryan (1954a, 1954b), while investigating the occasioiial finding of red cells sensitized to Cooinbs antiglobulin serum in ccrtaiii dogs with haemolytic anaemia due to phenylliydrazine administration, reported that hydrazinc in zjitro caused normal red cells either to agglutinate or to become agglutinablc by Coombs serum. Thus, a number of substances have been observed grossly to altcr red-cell stability, or, under certain conditions, to render the red-cell surface abnormally reactive with certain proteins. * This investigation was supported in part by a rescxch grant, P.H.S. No. G j ~ o 7 (C,) froin the National Institutes of Health, Public Health Service, and by a grant from the Helen Hay Whitney Foundation.

Increased Cell Membrane Permeability in the Pathogenesis of Hereditary Spherocytosis

The hemolytic process in hereditary spherocytosis (HS) is presumed to involve an intrinsic defect in the structure or metabolism of the red cell (1-3). The nature of the basic cellular defect remains enigmatic, although its manifestations are well characterized. Distinctive features of the HS red cell are its more spheroidal shape, small surface area, and high hemoglobin concentration and its exceptional proclivity toward splenic sequestration. However, the most singular feature of these cells is their abnormally rapid spheroidal change and increase in osmotic fragility on sterile incubation in vitro (4-6). This characteristic response of HS red cells has provided the most reliable method for diagnosis of the disorder (7). Efforts to identify a presumed biochemical lesion in HS red cells have had little success. A reported diminution in turnover of organic phosphate esters (8) has not been confirmed (9, 10), nor have abnormalities been found in the concentrations or kinetics of glycolytic intermediates or in glucose consumption of HS red cells (6, 11, 12). More recently, evidence has been presented (13, 14), but later denied (15, 16), that an abnormality exists in the phospholipids of the red cell membrane in this disease. With incubation, increased loss in lipids from spherocytic membranes has also been reported (17, 18). The general presumption in studies of HS red cells has been that a deficit existed in their energy metabolism. This presumption is difficult to reconcile with the findings by Harris and Prankerd (19) and Bertles (20) that the sodium efflux and influx, respectively, across HS cell membranes are

EFFECTS OF SULFHYDRYL INHIBITION ON RED BLOOD CELLS. II. STUDIES IN VIVO

Evidence is presented that the activity of the hexose monophosphate (HMP) pathway of red cells, assayed by r4C02 production from glucose-lJ4C, is regulated primarily by glutathione. Methemoglobin had little, if any, effect on the activity of this pathway. Increasing the ratio of oxidized to reduced glutathione, either by peroxidizing GSH to GSSG or by partially blocking GSH with NEM, increased the rate of HMP pathway metabolism. Complete blockage of cellular glutathione by NEM depressed the bulk of HMP pathway activity, despite little or no effect on the Embden-Meyerhof pathway. Sustained low levels of hydrogen peroxide, whether generated by aerobic oxidases or by the coupled oxidation of ascorbic acid with oxyhemoglobin, stimulated the HMP pathway of cells. This stimulation was potentiated by blocking catalase and was prevented by blocking GSH. The oxidation of NADPH by Hz02 in hemolysates was specifically dependent upon the presence of GSH. These results substantiate the existence in human red cells of the glutathione peroxidase mechanism proposed by Mills, whereby GSH protects cellular constituents such as hemoglobin from oxidative damage induced by HzOz. Oxidative denaturation of oxyhemoglobin to metand sulfhemoglobin by HpOz-generating mechanisms is markedly potentiated in cells lacking sufficient GSH, and commences only after GSH levels approach zero. In contrast, oxidationreduction catalysts such as methylene blue and acetylphenylhydrazine catalyze the direct oxidation of NADPH and hemoglobin, as well as GSH, by molecular oxygen. Although GSH is partially protective against these agents, they appear to damage red cells by virtue of their ability to bypass the GSH peroxidase mechanism and to cause oxidative injury despite persisting GSH.

Studies in vivo and in vitro on an abnormality in the metabolism of C3 in a patient with increased susceptibility to infection

In a patient with increased susceptibility to infection, lowered serum C3 concentration, and continuously circulating C3b, it was shown that purified (125)I-labeled C3 was converted to labeled C3b shortly after intravenous administration. The fractional catabolic rate of C3 was approximately five times normal at 10% of the plasma pool per hr. The synthesis rate and pool distribution of C3 were normal. Despite this evidence of C3 instability in vivo, no accelerated inactivation of C3 was found in vitro. Similarly, no free proteolytic activity could be detected in the patients serum, and serum concentrations of known protease inhibitors were normal.Complement-mediated functions, which were markedly deficient in the patients serum, could be restored partially or completely by the addition of a 5-6S heat-labile beta pseudoglobulin from normal serum. The C3 proinactivator, which has these physicochemical characteristics, was also shown to be either absent or nonfunctional in the patients serum. An unidentified 6S beta pseudoglobulin to which a monospecific antiserum was available was not detectable in the patients serum. This last protein appeared not to be a complement component, nor was it the C3 inactivator or proinactivator. Finally, the substance or substances necessary for the conversion of C3b to C3c were missing from the patients serum. The administration of 500 ml of normal plasma to the patient corrected all of his abnormalities partially or completely for as long as 17 days. The changes in C3 were dramatic; serum concentration rose from 8 to 70 mg/100 ml, and C3b could no longer be detected. A second metabolic study during this normalization period showed a decrease in fractional catabolic rate toward normal. The patients histamine excretion was constantly elevated but increased further after a warm shower and after receiving normal plasma; at both times he had urticaria. These observations were consistent with the endogenous production of C3a and the resulting histamine release from mast cells. The inactivating mechanism for C3a was apparently intact in the patients serum. The difference in the electrophoretic mobilities of C3b and C3c was shown as well as the electrophoretic heterogeneity of C3c. Suggestive evidence was also presented that the form of C3 with an activated combining site for red cells, previously postulated by others, is a transient C3 conversion product with an electrophoretic mobility slower than that of C3 on agarose electrophoresis.

A simple visual screening test for glucose-6-phosphate dehydrogenase deficiency employing ascorbate and cyanide.

HYPERSUSCEPTIBILITY to the hemolytic effects of such oxidant compounds as primaquine, acetylphenylhydrazine and sulfonamides may exist for a number of reasons, by far the most common of which is de...

The selective and conjoint loss of red cell lipids

The pattern of lipid loss from the membrane of red cells incubated in serum is influenced by the availability of glucose. Under homeostatic conditions with respect to glucose, cholesterol alone is lost. This results from esterification of free cholesterol in serum by the serum enzyme, lecithin:cholesterol acyltransferase, and is associated with a proportional decrease in membrane surface area, reflected by an increased osmotic fragility. This selective loss of membrane cholesterol also occurs in hereditary spherocytosis (HS) red cells, even after incubation for 65 hr in the presence of glucose. The loss of free cholesterol from red cells relative to its loss from serum, under these conditions, is greatest at higher hematocrits, similar to those found in the spleen. Although the selective loss of membrane cholesterol increases the spherodicity of normal red cells, it does not lead to a change in their rate of glucose consumption, and both the loss of cholesterol and the increase in osmotic fragility are reversible in vitro. Moreover, normal red cells made osmotically fragile by cholesterol depletion in vitro rapidly become osmotically normal and survive normally after their reinfusion in vivo.In contrast to this selective loss of membrane cholesterol, red cells incubated in the absence of glucose lose both cholesterol and phospholipid. This occurs more rapidly in HS than normal red cells and is followed by a disruption of cation gradients and then by hemolysis. Cholesterol and phospholipid lost under these conditions is not restored during subsequent incubations in vitro. Selective loss of membrane cholesterol is a physiologic event secondary to an altered state of serum lipids. It is reversible both in vitro and in vivo and neither influences cellular metabolism nor impairs viability. Conjoint loss of phospholipid and cholesterol, however, results from intrinsic injury to the red cell membrane which results from prolonged metabolic depletion.