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The objective was to assess clinical efficacy of 3 dosages of intravenous gammaglobulins to prevent infectious episodes in adult common variable immunodeficiency. We designed a randomized, double blind, dose-assessing study. The setting was at University Hospital, Out-patient Clinic. Our patients were twenty-one adult patients with common variable immunodeficiency. The measurements were comparative study of the number and severity of infections using 3 various dosages of intravenous gammaglobulins, each given monthly for M least 6 months. Results indicated four hundred and eighty-four infectious episodes occurred while giving 305 infusions of IVIG 200 mg/kg; 205 infectious episodes while giving 170 infusions of 400 mg/kg and 436 infectious episodes while giving 247 infusions of 600 mg/kg. The morbidity scores (infection/infusion) were 1.59, 1.21 and 1.77 respectively (p - N/S). There was no significant difference in the severity of infections on the above 3 dosages, and no difference in the duration of infection-free intervals. The conclusions resulted in no significant differences in morbidity in adult patients with common variable immunodeficiency treated in cross-over pattern with IVIG 200 mg/kg, 400 mg/kg and 600 mg/kg. Thus, high dosages of IVIG are not conferring better protection against infections in such patients.

Twelve adult patients with primary humoral immunodeficiency were treated for at least six months with IVIG 200 mg/kg/mo and then crossed over to a high dose of 600 mg/kg/mo. Polymorphonuclear and mononuclear cells of these patients were tested after the third infusion in the low-dose cycle and then after the third infusion in the high-dose cycle, each time a day before, four days after, and 14 days after intravenous infusion. Each time, patients' cells and normal cells were tested using normal sera and patients' sera. IVIG infusions led to a significant increase in the level of circulating IgG, which was much more prominent in the high-dose group. Phagocytosis, phagocytic index, intracellular bactericidal activity and chemotaxis of polymorphonuclear cells (PMNs) were at least as active as in healthy controls. Actually in both cycles patients' PMN's had slightly higher phagocytic activity than normal cells. Patients' serum in the high dose cycle supported chemotaxis better than normal serum. Efficient phagocytic activity was maintained throughout the cycle; however, it was more active (P < 0.0125) in the midcycle in the high-dose cycle. Superoxide generation was normal in all conditions. Monocytic function was also normal in all conditions tested. It may be concluded that as far as cellular phagocytic functions are concerned, the high dose of IVIG does not protect the host more efficiently than the low dose.

Synovial fluid PLA2 concentration was measured by an ELISA technique using monoclonal antibodies raised against human recombinant "synovial-type" group II phospholipase A2. This ELISA was specific for synovial-type PLA2 and did not detect pancreatic (group I) PLA2. In all synovial fluids examined, including rheumatoid, osteoarthritic, psoriatic, and gouty fluids, synovial fluid PLA2 enzyme activity significantly correlated with PLA2 immunoreactivity (P < 0.001). Within the limits of the ELISA technique, there was no evidence for the presence of specific or nonspecific modulation of PLA2 activity by either putative PLA2 activating or inhibitory proteins.

Phospholipase A2 (PLA2) activity was measured in the serum of 23 individuals infused intravenously with endotoxin (EN) at a dose of 4 ng/kg body weight. A marked increase in PLA2 was noted 3 h after EN challenge (mean 828 +/- 513 units/ml), reached its maximum at 24 h after the challenge (mean 2667 +/- 2442 units/ml), and was still evident at 48 h (mean 763 +/- 366 units/ml). In contrast, TNF levels were maximal (mean 712 +/- 375 pg/ml) 90 min after the EN challenge and subsided to very low values (5 +/- 5 pg/ml) 5 h after the challenge. There was a positive correlation between the maximum response of TNF and that of PLA2 (r = 0.82, P < 0.01). Administration of ibuprofen or pentoxifylline did not alter the PLA2 response. EN challenge did not affect serum pancreatic PLA2 concentration or that of the lysosomal cationic enzyme, lysozyme. Neutralizing antibody against human recombinant (synovial type) PLA2 completely abolished PLA2 activity in the sera tested. We conclude that EN infusions cause marked intravascular release of nonpancreatic secretory PLA2 and that the magnitude of this response seems to be related to the prior generation of TNF.

Soluble phospholipase A2 (PLA2) purified from rheumatoid synovial fluid (group II) and repurified Naja naja venom PLA2 (group I) were compared for their influence on phagocytic activity of human polymorphonuclear (PMN) and mononuclear (MO) phagocytes. Group II PLA2 reduced chemotaxis, adhesiveness, and intracellular bactericidal activity (ICBA) and induced release of muramidase from PMNs. Group I PLA2 suppressed chemotaxis, and enhanced ICBA but had no influence on other phagocytic functions. Group II PLA2 purified from synovial fluid or from placenta caused marked spontaneous superoxide generation followed by inhibition of phagocytosis-induced burst of energy. Group I Naja naja and porcine pancreatic PLA2 had no effect on superoxide generation. Group II but not group I PLA2 reduced markedly ICBA of monocytes. It may be concluded that human group II soluble PLA2, in concentrations comparable to those present in inflamed joints or in sera of patients with active arthritis or septic shock, causes spontaneous formation of the oxygen radical superoxide and release of lysosomal enzymes, and suppresses conventional phagocytic activities of PMNs and monocytes. Marked differences between group I and group II PLA2s may mean that these enzymes exert different influences on cell membrane.

Multiple forms of soluble phospholipase A2 (PLA2) are known to coexist in venoms of individual reptilian species. While similar observations in several mammalian species suggest that this is a common phenomenon, the functional implications are not yet understood. In attempting to devise therapeutic strategies for treatment of inflammatory disorders by inhibition of PLA2, it is imperative to define the various PLA2 species in the relevant compartments. Herein, we report the presence of three PLA2 isotypes in rheumatoid arthritis serum, one pancreatic and two nonpancreatic phospholipases A2. The pancreatic and one of the nonpancreatic forms were optimally active in 7 mM calcium at pH 7.5. The other nonpancreatic form was calcium-independent and optimally active at pH 7.0. Only the calcium-dependent nonpancreatic form was observed in rheumatoid synovial fluid. Of the three serum isotypes, only the calcium-dependent nonpancreatic form correlated with markers of disease activity, such as the joint count and Landsbury index. Therefore, not all soluble or circulating phospholipases A2 are relevant to inflammatory processes. Selective inhibition of the proinflammatory form of PLA2 may prove to have some therapeutic benefit while minimizing the possible adverse effects of this form of intervention.

Simultaneous assessment of the total number of bacteria (TNB) ingested, phagocytosis (Ph), phagocytic index (PI), and intracellular bactericidal activity (ICBA) of human monocytes was done by applying the fluorochrome acridine orange technique. Living bacteria stained orthochromatically green, whereas the dead ones were metachromatically red. The stain of extracellular bacteria was completely quenched by crystal violet counterstain. Using the Hypaque-Ficoll separation method combined with glass adherence, the yield of monocytes was 84 +/- 11%, the purity 90 +/- 8%, and the viability 99 +/- 1%. After 60 min of incubation of monocytes with Staphylococcus aureus, phagocytosis was 94 +/- 4%, PI 10.0 +/- 0.5, ICBA 76 +/- 5%, and TNB ingested 946 +/- 67/100 cells. E. coli B4 was equally ingested by PMNs and monocytes and killed intracellularly more efficiently by the latter type of cells. Over the ratios of bacteria to cells of 5:1 to 20:1, phagocytic activity of monocytes was equal or superior to that of PMNs. Phagocytic and bactericidal activities were enhanced by AB serum, more by the fresh one than by inactivated. Phagocytic activity of monocytes was markedly influenced by temperature of incubation. Room temperature (24 degrees C) significantly suppressed phagocytosis. Contrary to the previous beliefs no significant quantitative differences were found between phagocytic and bactericidal functions of monocytes as compared to polymorphonuclear phagocytes. The acridine orange-crystal violet method is simple, reliable, reproducible, and can be used for assessment of functional capacity of human phagocytes.

Seven cationic substances--human and egg-white lysozyme, RNase, protamine, histone, poly-L-lysine and poly-L-arginine; five cationic lysosomal fractions from human polymorphonuclears (PMNs); RNA; poly-L-glutamic acid; DNA; heparin; endotoxin; mastocytotropic agent compound 48/80; and cytochalasin B were tested for the influence on chemotaxis and random migration of human PMNs using under-agarose migration and Boyden chambers with two filters and [51Cr]PMNs. The above substances were either preincubated with PMNs, added to chemoattractants, or used instead of chemoattractants. In under-agarose migration method chemotaxis was inhibited by 11-35% when egg-white lysozyme, protamine, heparin, endotoxin, or compound 48/80 was added to the cells. High concentration of cytochalasin B inhibited chemotaxis by 73%. Cationic fractions I and V and low concentration of cytochalasin B enhanced chemotaxis by 11%, 41%, and 30%, respectively. When human and egg-white lysozyme, DNA, or cytochalasin B was added to the chemoattractants, motility of PMNs was inhibited. Cationic fractions II and V from human PMNs, when used as chemoattractants, enhanced cellular motility by 143-167%. Random migration was enhanced by heparin and inhibited by cytochalasin B and by cationic fractions from human PMNs. These findings suggest that various cationic and anionic substances and cationic fractions from human PMNs have heterogeneous influence on random migration and chemotactic activity of human PMN. Analysis relating chemotaxis to phagocytosis and to intracellular bactericidal activity (ICBA) has shown several patterns. Protamine, poly-L-lysine, poly-L-arginine, and agent compound 40/80 all inhibit chemotaxis and enhance phagocytosis and ICBA; cationic fractions II and V enhanced all three functions, whereas cytochalasin B suppressed phagocytosis and ICBA and had concentration-dependent modulatory influence on chemotaxis. It implies diverse mechanisms of action and possible impact on inflammatory reactions.

Monoclonal IgM cold agglutinins (CA) bind and, in the presence of complement, are cytotoxic to various mammalian cells. The impact of these autoantibodies on functional capacity of phagocytes has not been studied until now. Herein we report that sera with monoclonal IgM anti-I and anti-i CA significantly reduce adhesiveness, phagocytosis, phagocytic index, and intracellular bactericidal activity of human peripheral blood polymorphonuclear cells (PMNs) at 37 degrees C and 24 degrees C. Anti-i CA were more active than anti-I. Sera with monoclonal IgMs without CA activity reduced the total number of ingested bacteria but otherwise had no effect on phagocytic functions. There was no difference in the degree of inhibition when anti-i and anti-I CA were tested against cord, maternal, and adult PMNs. Chromatographically purified a-I and a-i CA inhibited markedly phagocytosis in concentrations as low as 1 mg/ml. Phagocytic activity of peripheral blood monocytes was inhibited by CA at 18 degrees C but not at 24 degrees C or 37 degrees C. Pepsin digestion or reduction and alkylation of chromatographically pure IgM CA abolished completely their inhibitory activity. Thus, in physiological temperatures, monoclonal IgM cold agglutinins impair various phagocytic functions of human phagocytes. It may add to the susceptibility to infections in patients in which such autoantibodies are synthesized.

Intravenous gamma-globulin was tested in a range of concentrations compatible with the increments obtained after therapeutic infusions for modulation of phagocytic functions of human polymorphonuclears (PMNs) and monocytes. Intravenous gammaglobulin in concentrations of 3.0 mg/ml or more increased adhesiveness and suppressed chemotaxis of PMNs. There was marked dose-dependent enhancement of opsonization of gram-positive and gram-negative microorganisms. Preincubation of PMNs with intravenous gamma-globulin caused enhancement of the total bacteria ingested, total bacteria killed, phagocytosis, and phagocytic index, when gram-positive and gram-negative bacteria were tested. During phagocytosis, there was no release of LDH or lysozyme; however, there was release of beta-glucuronidase. No significant difference in phagocytic enhancement was found when filtered and native intravenous gamma-globulin preparations were compared. There was marked enhancement of the superoxide anion generation by intravenous gamma-globulin above the concentration of 0.01 mg/ml. Intravenous gamma-globulin also markedly enhanced phagocytic activity of monocytes. Therefore, intravenous gamma-globulin modulates not only opsonization-related phenomena, but also exerts a complex influence on other aspects of phagocytic activity.

Cationic fraction III from the lysosomes of normal human peripheral blood polymorphonuclear cells (PMNs) was found to contain superoxide generation enhancing protein (SGEP). Herein, we report on the influence of partially purified SGEP obtained from fraction III (subfractions III-5 and III-6), on various phagocytic functions of human PMNs. SGEP markedly enhanced intracellular bactericidal activity of human peripheral PMNs. The enhancement was time and dose dependent. It also reduced adhesiveness of the PMNs. SGEP did not influence chemotaxis, phagocytosis or phagocytic index. These findings are compatible with our original observation regarding superoxide generation enhancement properties of SGEP.

The cationic proteins from neutrophil lysosomes have been shown to modulate phagocytic activity of granulocytes. The present study reports the effects of the cationic protein fractions on the generation of O2- by human PMNs during phagocytosis. Human PMNs were reacted with different phagocytic stimuli in the presence and absence of lysosomal cationic proteins and the amount of O2- generated was determined by superoxide dismutase inhibitable reduction of cytochrome c. Total cationic protein extract from neutrophil lysosomes enhanced O2- generated by PMNs during the phagocytosis of IgG-coated latex beads and opsonized zymosan particles. The analysis of the fractions of cationic proteins obtained from a Sephadex G-75 column showed that the O2- generation-enhancing activity was associated with the proteins eluted in fractions III and IV. A protein fraction mainly eluted in void volume inhibited the cytochrome c reduction by O2- formed during phagocytosis. This was due to the presence of superoxide dismutase-like activity since O2- generated by the xanthine-xanthine oxidase system was also inhibited by this fraction. The cationic protein fractions III and IV from the Sephadex G-75 column were further subfractionated. Although the O2(-)-enhancing activity was eluted in the same fractions as chymotrypsin activity, there was no quantitative correlation between the amount of O2- generation and chymotrypsin activity. Moreover, commercial chymotrypsin did not enhance O2- generation. Electrophoretic analysis of the isolated protein fractions suggests that O2- generation enhancing protein (SGEP) is different from lysozyme or chymotrypsin and probably represents previously undescribed protein.

Cationic lysosomal proteins from human polymorphonuclears (PMN) were isolated by column chromatography and divided into five fractions. On acrylamide gel electrophoresis, fraction I had four bands slower than lysozyme (LZM) mobility; fraction II had five or six bands slower than LZM; fraction III had at least seven bands slower and two bands faster than LZM; fraction IV contained LZM, two bands faster and a few faint bands slower than LZM; fraction V was composed of almost pure LZM. Partial characterization of the fractions showed presence of neutral protease in fractions I-IV, chymotrypsin in fraction III, lysozyme in fractions IV and V, and phospholipase A2 mainly in fractions II and III. Modulatory activity of fractions I-V were tested at concentrations up to 50 micrograms/ml. Enhancement of phagocytosis of Staphylococcus aureus was observed by fractions I, IV, and V, whereas phagocytic index was enhanced by all but the fraction II. Intracellular bactericidal activity (ICBA) was markedly enhanced by fractions I, II, and V. Addition of DNA or cytochalasine B inhibited or abolished phagocytosis-enhancing activity of cationic fractions. Their influence on ICBA was much less pronounced. Fraction III enhanced phagocytic index and phagocytosis of E. coli, whereas fractions I and II enhanced intracellular bactericidal activity against this bacteria. Enhancement of phagocytic activity of monocytes has also been observed. The data suggest that some cationic lysosomal fractions from human PMNs enhance phagocytosis and phagocytic index by human PMNs and monocytes and intracellular bactericidal activity of human PMNs. This alternative pathway of phagocytic enhancement is unrelated to the previously described enhancers of phagocytosis and may play a role in defense mechanisms against infection.

The influence of plasma proteins on quantitation of the proinflammatory enzyme phospholipase A2 (PLA2) from rheumatoid synovial fluid was studied using two different assays. Human and bovine serum albumins increased the rate of PLA2 hydrolysis of membrane-associated phospholipid substrates. In contrast, albumin profoundly inhibited the PLA2 hydrolysis of synthetic phospholipids in micellar dispersion. Other plasma proteins (alpha, beta and gamma-globulins) had minimal effect on PLA2 activity in either assay system. Since the presence of albumin may compromise estimation of PLA2 and of phospholipase-inhibitory proteins, the appropriate selection of assay conditions is obligatory for the accurate quantitation of their respective activities.