Phagocytosis in subacute bacterial endocarditis. Localization of the primary opsonic site to Fc fragment.

The opsonic properties of immune gammaG-globulins isolated from patients with chronic septicemic conditions, principally subacute bacterial endocarditis were studied. Opsonic capacity as well as complement-fixing properties of gamma-globulins appeared to be closely associated with integrity of Fc structures. Progressive pepsin digestion of immune gammaG-globulins, as monitored by successive loss of Gm(a) and Gm(b) antigens, abolished opsonic activity. Colostral gammaA, containing agglutinating antibacterial antibodies but no demonstrable complement-fixing activity, was devoid of opsonic capacity. Reduction of gamma-globulin opsonins with 0.01 or 0.1 M mercaptoethanol progressively abolished opsonic activity in parallel with loss of ability of treated gamma-globulins to fix complement with bacteria. Treatment of gamma-globulin opsonins with 0.01 M sodium metaperiodate also produced complete loss of opsonic capacity in parallel with loss of Gm(b) Fc antigens. These findings, together with antiopsonic effects demonstrable with anti-gamma-globulin factors showing primary reactivity with Fc structures, indicate that the opsonic property of immune gamma-globulins requires the participation of structures integral to the Fc region of gamma-globulin.

Indirect visualization of Staphylococcus aureus protein A.

Specific but nonimmunologic reaction between staphylococcal protein A and the Fc portion of gamma globulin provided the basis for ultrastructural studies to determine the localization of protein A, using intact staphylococci and labeled myeloma gamma G-globulin. Protein A appeared to be part of the outermost layer of the staphylococcal cell wall. Strains with protein A demonstrated a coating of myeloma globulin over the entire bacterial surface. There was no coating of strains without protein A. Identification of protein A on the surface of the staphylococcal cell wall provides evidence that this may be the first material in contact with host environment. It probably accounts for apparent cross-reactions of staphylococci with antibodies to many antigens. More importantly, even in the nonimmune host protein A immunoglobulin reactivity may initiate complement activation and inflammatory reactions including chemotaxis and pus formation.

X-linked inheritance in females with chronic granulomatous disease.

Chronic granulomatous disease in males is familial and its transmission is is usually clearly x-linked. The mode of inheritance in females with the syndrome is unknown and the carrier state difficult to identify. Defective polymorphonuclear leukocyte bactericidal activity in this disease is associated with an absence of the respiratory burst generated in stimulated phagocytes and may be detected by the chemiluminescence assay. Polymorphonuclear leukocytes from three of four females with chronic granulomatous disease had extremely low chemiluminescence production, their asymptomatic mothers had intermediate values, and their fathers were normal. Polymorphonuclear neutrophils of two affected males in these kinships generated no chemiluminescence, whereas two of seven female relatives had intermediate values, and all nonaffected males had normal values. In the three families in which leukocytes were studied by nitroblue tetrazolium reduction, two populations of neutrophils were demonstrated for the female patients and/or their mothers. The wide phenotypic variability for clinical disease, evidence of two leukocyte populations in the patients or their mothers, and low but detectable leukocyte chemiluminescence in the affected females is consistent with the Lyon hypothesis of x-chromosome inactivation in these families. The findings suggest an x-linked inheritance in these females with chronic granulomatous disease.

Hyperactivity of neutrophil leukotactic responses during active bacterial infection.

To determine if changes in neutrophil leukocyte function occur during active bacterial infection, the neutrophils of 25 patients with active bacterial infection and 25 age-matched controls were compared for leukotactic activity, random mobility, and nitroblue tetrazolium reduction. The neutrophil leukocytes of patients with bacterial infection were hyperactive in unidirectional movement toward a chemotactic stimulus as measured in the leukotactic assay and usually had increased nitroblue tetrazolium reduction. The mean leukotactic index was 165+/-56 in patients with bacterial infection and 70+/-11 in controls (P < 0.001). After 7-10 days of appropriate therapy with clinical and bacteriological response, leukotactic activity returned to normal values. A hyperactive leukotactic response continued, however, in patients with persisting bacterial infection. The hyperactive leukotactic response of circulating neutrophils appears to be an early and sensitive event in the inflammatory cycle stimulated by bacterial infection and may aid in the localization of invading bacteria.

In vitro bactericidal capacity of human polymorphonuclear leukocytes: diminished activity in chronic granulomatous disease of childhood.

Diminished bactericidal capacity was found to be characteristic of polymorphonuclear leukocytes (PMN) from five children with the clinical syndrome of granulomatous disease of childhood. The PMN from these children demonstrated nearly normal phagocytic capacity, and the majority of viable bacteria, after 2 hours of incubation in the phagocytosis system, were found associated with leukocytes. The morphology of the unstimulated polymorphonuclear leukocytes from patients with chronic granulomatous disease was similar to those from normal persons of similar ages by light and electron microscopy. In addition, the total lysozyme and phagocytin activity of leukocyte extracts from these patients was similar to those from equal numbers of leukocytes from controls.A striking difference in the cytoplasmic response after phagocytosis characterized the PMN of the patients with granulomatous disease. Whereas degranulation, vacuole formation, and rapid bacterial digestion were the rule in the PMN from controls, little degranulation and persistence of intact bacteria in the cytoplasm characterized disease. The deficiency of bactericidal capacity and the minimal degranulation after active phagocytosis by the PMN of these children with an inherited syndrome suggest that separate metabolic processes are involved in phagocytosis and in intracellular digestion. Continuing study of the metabolic function of leukocytes from these children should provide an opportunity for increased understanding of the metabolic basis for degranulation and intracellular digestion in phagocytic cells.

Serum opsonin, bacteria, and polymorphonuclear leukocyte interactions in subacute bacterial endocarditis. Anti-gamma-globulin factors and their interaction with specific opsonins.

The effect of anti-gamma-globulin factors on 7S gamma-globulin opsonins from patients with subacute bacterial endocarditis has been examined with a quantitative in vitro phagocytosis system. Human anti-gamma-globulin factors from patients with subacute bacterial endocarditis and rheumatoid arthritis inhibited the opsonic action of 7S gamma-globulin specifically bound to bacteria. A similar antiopsonic effect was obtained with rabbit antiserum to human gammaG globulin. The antiopsonic effect of anti-gamma-globulin factors did not correlate with their ability to potentiate agglutination of bacteria by 7S antibody. Competition was demonstrated between the antiopsonic effect of anti-gamma-globulin factors and the phagocytosis-promoting action of heat-labile serum factors containing hemolytically active complement.

The pattern of genetic transmission of the leukocyte defect in fatal granulomatous disease of childhood.

The leukocyte-phagocytic function test which was found to be abnormal in boys with fatal granulomatous disease of childhood has been found to be abnormal to an intermediate extent in their mothers. Nine of nine mothers were shown to be abnormal, whereas none of eight fathers and none of five healthy brothers exhibited a defect. 10 of 16 female siblings were abnormal to the same degree as their mothers, as were all three maternal grandmothers available for study. Assuming that this intermediate functional defect represents the heterozygous state, the nine family pedigrees are entirely compatible with the concept that the trait is transmitted on the X-chromosome.A tetrazolium dye-phagocytosis histochemical test was also abnormal in the carrier females and provided independent confirmation of the selection of the female siblings suspected of being carriers for the trait. In addition, this procedure gives indirect evidence that the gene in question is subject to the random inactivation that appears to affect many X-linked genes in mammalian females. The family members were also studied with two of the metabolic assays that have been shown to be abnormal in the cells of affected boys. One assay, the oxidation of the first carbon of glucose-1-(14)C by the isolated leukocytes, was significantly abnormal in the cells of carrier females. The other assay, the oxidation of formate-(14)C by leukocytes of heterozygotes was not significantly different from control values. The practical problem of diagnosing patients would appear to be best solved with a tetrazolium dye procedure, whereas the more subtle abnormality in carrier females is best detected with the leukocyte function test. Improved methods for the function test are being developed.

Human alveolar macrophage cytophilic immunoglobulin G-mediated phagocytosis of protein A-positive staphylococci.

Human alveolar macrophages (AM) have recently been reported to ingest and kill a strain of Staphylococcus (502A) in the absence of opsonins. To further investigate the mechanism of non-opsonic recognition, we studied phagocytosis of 23 clinical and laboratory strains of S. aureus and Staphylococcus epidermidis by AM, and by blood polymorphonuclear leukocytes (PMN) and monocytes (MN). In the absence of opsonins, AM phagocytized 18 protein A-positive but not 5 protein A-negative strains of staphylococci, and the efficiency of phagocytosis directly correlated with the amount of protein A present in the bacterial cell wall (r = 0.86, P less than 0.001). Furthermore, AM rosetted around protein A-coated Sepharose beads, but not around beads without protein A. In contrast, PMN did not phagocytize nonopsonized staphylococci, and did not rosette around either type of Sepharose. MN phagocytized protein A-positive staphylococci, but much less efficiently than AM, and showed some rosetting around protein A-coated Sepharose. The nature of the AM receptor for protein A-positive staphylococci was studied. The surface of AM was positively stained with fluorescein-conjugated antibody to human IgG, but not with IgA- or IgM-specific conjugates. No such surface-immunoglobulins were detected on PMN, and MN were only weakly positive for surface IgG. Pretreatment of AM with F(ab')2 fragments specific for human IgG (anti-Fc) inhibited subsequent phagocytosis of protein A-positive staphylococci. There was no evidence that the AM surface IgG was aggregated or immunecomplexed. From these studies we conclude that human AM possess cytophilic IgG antibodies, which can function as receptors for phagocytosis of protein A-positive staphylococci.

The key role of peptidoglycan in the opsonization of Staphylococcus aureus.

In an effort to determine the staphylococcal cell surface component(s) of importance in opsonization, cell walls (peptidoglycan and teichoic acid) and peptidoglycan were isolated from Staphylococcus aureus strain H grown in [3H]glycine-containing broth. After incubation of the cell walls and peptidoglycan with various opsonic sources, uptake by human polymorphonuclear leukocytes was measured. The opsonic requirements for phagocytosis of cell walls and peptidoglycan were found to be similar to those of intact bacteria. Removal of teichoic acid from the cell wall did not affect opsonization. Likewise, a teichoic acid-deficient mutant strain of S. aureus H was opsonized in a manner similar to that of the parent strain. Immunoglobulin G functioned as the major heat-stable opsonic factor and both the classical and alternative pathways participated in opsonization. Kinetic studies revealed that opsonization of peptidoglycan, as well as C3-C9 consumption by peptidoglycan, proceeded at a slower rate via the alternative pathway (C2-deficient serum) than when the classical pathway was present (normal serum). The ability of peptidoglycan to activate C3-C9 was significantly reduced when normal and C2-deficient sera were preabsorbed with peptidoglycan at 2 degrees C suggesting that antibodies to peptidoglycan may be involved in activation of both the classical and alternative complement pathways. Thus, peptidoglycan appears to be the key cell wall component involved in staphylococcal opsonization, and it is suggested that host response to peptidoglycan, a major cell wall component of most gram-positive bacteria, may be related to the development of "natural immunity" to this group of microorganisms.

The chemiluminescence response of human platelets.

Human platelets and platelet particulate fractions were found to emit a burst of chemiluminescence during incubation with arachidonic acid. The magnitude of light emission was directly related to the number of platelets in the reaction mixture and varied little for the same individual from day to day. The chemiluminescence response of platelets was localized to the particulate fraction and was almost totally oxygen dependent. In addition to arachidonate, seven other polyunsaturated fatty acids, including several that are not prostaglandin precursors, also induced platelet chemiluminescence.A correlation was sought between chemiluminescence and platelet prostaglandin synthesis. Platelets incubated in low concentrations of aspirin, or platelets from subjects who had ingested aspirin, had markedly decreased arachidonic acid-induced chemiluminescence. Salicylic and sulfosalicylic acid had no inhibitory effect. A time-response curve of aspirin inhibition of arachidonate-induced chemiluminescence closely paralleled a time-response curve of aspirin inhibition of malondialdehyde production. Linoleic acid-induced platelet chemiluminescence was also markedly inhibited using aspirin-incubated platelets or platelets from subjects who had ingested aspirin. These studies implicate activation of the enzyme prostaglandin synthetase in the arachidonate-induced platelet chemiluminescence. They provide evidence that linoleic acid may also specifically activate platelet cyclooxygenase to produce electronically excited species capable of light emission.

Potentiation of opsonization and phagocytosis of Streptococcus pyogenes following growth in the presence of clindamycin.

Streptococcus pyogenes, bearing M-protein on its surface, resists opsonization by normal human serum and subsequent phagocytosis by human polymorphonuclear leukocytes. Previous studies have shown that M-protein positive organisms are poorly opsonized by the alternate pathway of complement. In an attempt to define further the role of the surface components of S. pyogenes in this process, we examined the ability of clindamycin, an antibiotic that inhibits protein biosynthesis, to alter bacterial opsonization. An M-protein positive strain of S. pyogenes was grown in varying concentrations of clindamycin at levels lower than those which inhibited growth, i.e., at levels less than the minimal inhibitory concentration. These bacteria were incubated with purified human polymorphonuclear leukocytes and peripheral blood monocytes. Significant enhancement of bacterial opsonization, phagocytosis, and killing resulted. Measurement of complement consumption and binding of the third component of complement (C3) onto the bacterial surface demonstrated that organisms grown in the presence of clindamycin activated complement more readily and fixed more C3 on their surface. Electron microscopy revealed the probable basis for these findings. Streptococci exposed to clindamycin during growth were largely denuded of surface "fuzz," the hairlike structures bearing M-protein. We conclude that the incorporation of clindamycin at concentrations that fail to inhibit growth of S. pyogenes nevertheless causes significant changes in the capacity of these bacteria to resist opsonization by serum complement. These findings support the hypothesis that M-protein inhibits bacterial opsonization by interfering with effective complement activation on the bacterial surface.

Opsonin-independent phagocytosis of surface-adherent bacteria by human alveolar macrophages.

Opsonin-independent mechanisms of phagocytosis may be important in host defense of certain body sites such as the lung. In this study, one such mechanism, "surface phagocytosis," was investigated by measuring the uptake of unopsonized [3H]-labeled Staphylococcus aureus and Pseudomonas aeruginosa adherent to a plastic surface by human alveolar macrophages (AM) and peripheral blood polymorphonuclear leukocytes (PMN). Efficient uptake of unopsonized bacteria by both cell types was observed. Electron microscopic studies suggested that the manner in which these cell types encounter adherent bacteria is different. While AM appear to gather in organisms at their periphery as they spread out upon the underlying substrate, PMN seem to sweep bacteria up as they move along the plastic surface. Bacterial killing determined by a fluorochrome microassay was decreased by AM compared to PMN. Although the precise mechanism whereby phagocytes recognize unopsonized bacteria adherent to a surface remains to be determined, this aspect of phagocytic cell function may prove to have clinical relevance.

Kinetics of staphylococcal opsonization, attachment, ingestion and killing by human polymorphonuclear leukocytes: a quantitative assay using [3H]thymidine labeled bacteria.

A method has been developed for studying quantitatively the separate processes of bacterial opsonization, phagocytosis, and killing by human polymorphonuclear leukocytes using [3H]thymidine labeled Staphylococcus aureus. Phagocytosis is determined by assaying for leukocytes-associated radioactivity after differential centrifugation and washing the leukocytes. Opsonization is studied by incubating bacteria with an opsonic source for varying durations and then adding leukocytes. By treatment of samples with the muralytic enzyme, lysostaphin, the attachment and ingestion phases of phagocytosis can be separated. Sampling for colony forming units after disruption of the leukocytes permits the measurement of bacterial killing. Using this method, differences in the kinetics of staphylococcal opsonization by normal and C2 deficient sera were defined, opsonic influences on the attachment and ingestion phases of pH agocytosis were delineated, and the influences of different opsonins and leukocyte populations on killing were determined.

An enzyme-linked immunoassay (ELISA) for measurement of lactoferrin.

An enzyme-linked immunosorbent assay has been developed for quantitation of lactoferrin (LF) in body fluids. An indirect double-sandwich method was used which allows a sensitivity of 3 ng LF/ml in samples of polymorphonuclear cell lysates and serum. Mean LF content of serum was 0.307 +/- 0.066 micrograms/ml (n = 18). Mean LF content of polymorphonuclear cells was 4.90 +/- 1.48 micrograms/10(6) PMN. Concentrations of LF were similar in serum and in plasma of EDTA anticoagulated blood. Advantages of this method include its rapidity, and radioactivity is not required.

Phagocytosis by polymorphonuclear leukocytes of Staphylococcus aureus and Pseudomonas aeruginosa adherent to plastic, agar, or glass.

Phagocytic cells may encounter bacteria in vivo that are stationary or adherent to a surface. In this study, recently developed in vitro techniques were adapted to evaluate the interaction of polymorphonuclear leukocytes (PMN) with adherent Staphylococcus aureus and Pseudomonas aeruginosa. By measuring the uptake of radiolabeled bacteria, we found that normal human PMN readily phagocytize these organisms when they are attached to plastic or when they are grown on the surface of nutrient agar. Bacteria adherent to glass elicited a chemiluminescent response, and such organisms were phagocytized and killed by PMN. Opsonization of S. aureus and P. aeruginosa was not required for surface phagocytosis, chemiluminescence, or killing. These new methods should allow evaluation of certain biological and clinical aspects of surface phagocytosis in host defense.

The role of complement, immunoglobulin and bacterial antigen in coagulase-negative staphylococcal shunt nephritis.

We describe three patients with arrested hydrocephalus in whom glomerulonephritis developed secondary to Staphylococcus epidermidis bacteremia from an infected ventriculoatrial shunt. Investigation of the immune-mediated renal disease associated with this chronic infection showed that (1) complement depletion during the acute phase of bacteremia and nephritis was predominantly via the classic pathway; (2) rheumatoid factor was associated with bacteremia, fever, proteinuria and low complement levels; (3) early complement components (C1q, C4, C3), immunoglobulin (predominantly immunoglobulin M [IgM], Staph. epidermidis antigen(s) and electron denxe subendothelial deposits were localized within the renal glomerulus; (4) C1q, and IgM derived from patient serums, were the most prominent in vitro immunoreactants to Staph. epidermidis cell walls; and (5) the causative organisms, Staph. epidermidis, shared common antigens with Staph. aureus, and antibody from patient serums cross reacted with extracts from both of these organisms.

Bactericidal and metabolic function of polymorphonuclear leukocytes.

The bactericidal and metabolic function of the phagocytic system requires integration of several complex humoral and cellular factors responding to different regulators. Polymorphonuclear leukocytes are highly mobile cells, capable of phagocytosis of bacteria or fungi with formation of a "cellular digestive system" containing reactive oxygen radicals, hydrogen ions, and digestive enzymes. The unique metabolism of oxygen in neutrophils results in release of energy as light (chemiluminescence) a response closely associated with microbiol killing. Neonatal neutrophils cope with normal bacterial challenges in vitro as efficiently as adult neutrophils; however, these cells have decreased capacity for locomotion, decreased deformability, decreased phagocytosis in low serum concentrations, and decreased chemiluminescence. These subtle defects in function can be amplified by exaggerated challenge which may be related to a higher incidence of sepsis during the neonatal period.

The chemiluminescence response and bactericidal activity of polymorphonuclear neutrophils from newborns and their mothers.

Chemiluminescence (CL) was measured in the polymorphonuclear neutrophils (PMN) of 18 normal term infants, their mothers, and controls during phagocytosis of opsonized zymosan particles. Chemiluminescence was significantly lower in the PMN of newborns in comparison with the PMN of their mothers and of the controls. Depressed bactericidal activity was demonstrated in newborn PMN, in comparison with the activity of the PMN of their mothers and controls, when challenged with Escherichia coli at large bacteria-PMN ratios. Uptake of radio-labeled bacteria by PMN was identical in newborns, mothers, and controls, which indicates that reduced CL was not a result of impaired ingestion. Therefore, PMN of normal term infants have both depressed oxidative metabolic responsiveness as measured by CL and depressed bactericidal capacity.

The microbiology of serous and mucoid otitis media.

One hundred forty-four serous and mucoid effusions were cultured for aerobic bacteria, Mycoplasma pneumoniae, and virus. Thirty percent of all effusions yielded an unequivocally positive culture for aerobic bacteria. Although serous effusions were culture positive as often as mucoid effusions, Haemophilus influenzae was isolated predominantly from serous effusions and Staphylococcus epidermidis predominantly from mucoid samples. Only one of 73 effusions yielded a viral isolate (Herpesvirus hominis). None of 33 effusions yielded M pneumoniae, and only one of 17 effusions yielded an anaerobe (Propionibacterium). These findings suggest that aerobic bacteria may play a role in the pathogensis of serous and mucoid otitis media.

Leukocyte granulation abnormality associated with normal neutrophil function and neurologic impairment.

This article describes studies of two unrelated patients, ages 5.5 and 26 years, with leukocyte granulation abnormalities similar to those in the Chediak-Higashi syndrome. Both patients presented with neurologic manifestations characterized by psychomotor impairment, but neither had any evidence of oculocutaneous albinism, photophobia, or increased susceptibility to pyogenic infection. The leukocytes were studied for cytochemical, ultrastructural, ultrastructural cytochemical, and functional characteristics. Abnormal granules were present in neutrophils, eosinophils, basophils, monocytes, and lymphocytes; in the neutrophil series the abnormalities involved both the azurophilic and specific granules. On ultrastructural examination, the abnormal granules in the neutrophils were found to result from fusion of both peroxidase-positive and peroxidase-negative granules. Large numbers of normal granules were also present. The abnormal large granules in the eosinophils and basophils were the result of fusion of normal granules. The neutrophil function studies showed normal chemotaxis, chemiluminescence, bactericidal activity, and nitro-blue tetrazolium reduction. The normal neutrophil function studies were paralleled by the clinical histories in that neither patient had a history of severe infectious episodes.