Opsonins and erythrophagocytosis

The author studied the capacity of granulocytes and reticular cells of rat bone marrow to phagocytize mouse red blood cells in the action of opsonins against them. A comparative assessment of phagocytosis of fresh and formaldehyde-treated mouse red blood cells was conducted. A moderate phagocytosis observed with the use of fresh erythrocytes and immune noninactivated rat serum was less pronounced when inactivated serum was used. No phagocytosis occurred with rat nonimmune serum. On the contrary, when formaldehyde-treated erythrocytes were used with the same sera phagocytosis was much stronger.     

Opsonic effect of antiserum and complement on phagocytosis by macrophages from the peritoneal cavity of the Japanese eel, Anguilla japonica

Macrophage exudates in the Japanese eel, Anguillu japonica, were induced by intraperitoneal injection with a mixture of Edwnrdsiella bacterin, proteose peptone and liquid paraffin, and the opsonic effect of antiserum and complement on the phagocytic activity of the macrophages was studied.
The macrophages contained a round nucleus with a nucleolus, and an agranular cytoplasm with projecting processes. These cells showed phagocytosis of sheep red blood cells (SRBC). An opsonic effect of antiserum was recognized in terms of a significant increase of macrophage phagocytic activity against SRBC treated with antiserum relative to that against SRBC treated with inactivated normal serum. Complement, however, did not enhance the phagocytic activity of macrophages.     

The method for estimation of opsonic activity in sera of infants

Three methods for the estimation of opsonic activity in the sera of newborn children were tested. Two of them, based on the phagocytosis of opsonised bacteria labelled with radioactive phosphorus³²P as measured byin vivo blood clearance or uptake of bacteria in perfused isolated liver, were found to be unsuitable for long term dynamic study mainly because they do not permit the testing of series of samples. The third method (using isolated phagocytic cellsin vitro) permits the differentiation of the opsonic effect of complement and antibody and, furthermore, the firmness of the bond between microbes and phagocytes (which reflects the degree of opsonization) can be established. It was found that a 2-mercaptoethanol-resistant antibody, probably of the IgG type, was responsible for the opsonic activity of children's sera toEscherichia coli 083. Homologous antibody (toEscherichia coli 083) could be differentiated from beterologous antibody (toEscherichia coli 086) using the opsonic test only at low dilutions of sera. The combination of newborn piglet complement and antibody of children's sera yielded higher values of opsonic activity than either component separately.     

Enhancement of phagocytosis of parasitized and nonparasitized red cells in acute Plasmodium berghei malaria

The phagocytosis by mouse peritoneal macrophages of parasitized red cells (PRCs) and nonparasitized red cells (nonPRCs) separated from Plasmodium berghei infected blood was studied in vitro. Peritoneal macrophages from acutely infected mice and normal mice were cultured on coverslips. PRCs and nonPRCs were fractionated by Ficoll density gradient centrifugation from Plasmodium berghei infected blood. PRCs were fed in triplicate cultures to normal macrophages in normal serum, normal macrophages in acute serum, acute macrophages in normal serum, and acute macrophages in acute serum. Similarly, nonPRCs were fed to macrophages in the same combinations of normal and acute macrophages and serum. In acute serum, acute macrophages ingest PRCs significantly more readily than normal macrophages (p less than 0.05). Acute macrophages in acute serum ingest PRCs more readily than acute macrophages in normal serum, but the difference does not achieve statistical significance. Acute serum apparently decreases the phagocytosis of PRCs and nonPRCs by normal macrophages, perhaps because of an inhibitory antibody present in acute serum. As with PRCs, in the presence of acute serum, acute macrophages ingest nonPRCs significantly more readily than normal macrophages (p less than 0.05).     

Fasciola hepatica in rats: transfer of immunity by serum and cells from infected to F. hepatica naive animals

Immune, hyperimmune, and nonimmune serum samples were collected from inbred rats following 10 to 15 weeks of one [5 metacercariae (mc)/rat], two (5 mc followed by 30 mc/rat) or no (uninfected) exposure to Fasciola hepatica. Lymphoid cells also were collected from these donors. Inbred, naive rats in groups receiving immune serum, hyperimmune serum, nonimmune serum (serum control), immune cells, hyperimmune cells, and nonimmune cells (cell control) received intraperitoneally either a total of 20 ml of serum or a total of 3 x 10(8) viable lymphoid cells. A challenge infection of 30 mc/rat was administered orally at about the time of serum or cell transfer. The transfer of immunity was evaluated by examining recipient rats for parasites 4 and 8 weeks after challenge. Some hematological parameters and the precipitating antibody response of the recipients were monitored also. Hyperimmune serum, unlike immune serum, consistently provided a significant degree of protection in recipient rats. The precipitating antibody titre of this serum was higher than that obtained from the immune donor group. The importance of a second sensitization to obtain sufficiently potent serum was demonstrated. Lymphoid cells from infected donors did not consistently confer protection on recipients. Thus, the expression of protective immunity against F. hepatica seemed to be more dependent on the presence of antibodies than on cells. The hematological parameters of the recipients, in general, supported this observation. The precipitating-antibody response of protected rats was lower than that of unprotected animals following challenge, presumably because the development of fewer worms in the former provided less antigenic stimulation.     

Red cell ageing: phagocytosis and life-span of young and old erythrocytes fractionated by centrifugation

Young and old red blood cells, separated by centrifugation on the basis of differences in cell density, were submitted to phagocytosis by either autologous human alveolar macrophages or syngeneic murine bone-marrow macrophages. Young cells adhere to macrophages, but to a much smaller extent than old ones. The influence of both type and quality of the separation procedure on the differences observed between the two erythrocyte subpopulations is discussed in the light of the half-life times of murine young and old red blood cells. Fractionation according to age was obtained following the method of Murphy (1973) and glutamate oxalo-acetate transaminase activity was measured and used as an indicator of both cell age and separation.     

Differential properties of organ-specific serum opsonins for liver and spleen macrophages

Earlier we reported that serum contains organ-specific opsonins which selectively enhance recognition of liposomes by macrophages in the specific organs of the reticuloendothelial system (Moghimi, S.M. and Patel, H.M. (1988) FEBS Lett. 233, 143-147). The results presented here describe the properties of these organ-specific opsonins which differentiate between liver-specific and spleen-specific opsonins responsible for the enhancement of phagocytosis of liposomes by Kupffer cells and spleen macrophage, respectively. Liver-specific opsonin is a heat-stable macromolecule which on heating or on freezing and thawing exhibits enhanced opsonic activity. Serum also contains a dialysable factor which inhibits its opsonic activity. On the other hand, the spleen-specific opsonin is a heat-labile macromolecule which is sensitive to freezing and thawing and requires a dialysable serum co-factor for its optimum opsonic activity on spleen macrophages. Removal of this factor from serum brings about an irreversible conformational change in the opsonin. Evidence suggests that the spleen-specific opsonin may be composed of more than one different opsonin molecule. It is suggested that the serum factor(s) that inhibits liver-specific opsonic activity and enhances the spleen-specific activity may not be the same molecule, but in both the cases the factor(s) may mediate its function by modifying the process of the opsonisation of liposomes or by influencing the interaction of the opsonised liposomes with the respective cells. We propose that purification of the organ-specific opsonins may provide an opportunity to target drug carriers selectively to a specific organ of the reticuloendothelial system, and help us to evaluate their role in the altered opsonin states known to exist in certain diseases.     

Interaction of C-reactive protein with lymphocytes and monocytes: complement-dependent adherence and phagocytosis.

The serum constituent C-reactive protein (CRP), which activates the classical complement (C) pathway when reacting with its substrates, was examined for its ability to mediate reactions of opsonic adherence and phagocytosis. Erythrocytes coated with C-polysaccharide (CPS) and reacted with CRP (E. CPS-CRP) failed to adhere to B cells and displayed only minimal adherence to monocytes. However, upon the addition of absorbed C or purified C components these cells were found to possess the cleavage products C4b and C3b, which in turn resulted in attachment of these cells to both human B lymphocytes and peripheral blood monocytes. E. CPS-CRP treated with C in the absence of antibody were readily phagocytosized by glass-adherent human monocytes. The phagocytosis of E. CPS-CRP-C was not only mediated by CRP but also required the presence of CRP on the surface of the red cells. The extent of ingestion was proportional to the amount of CRP on the red cell intermediate and was reduced by blocking monocyte receptors with aggregated human gamma-globulin (HGG) at concentrations which did not impair the uptake of other particles. The mediation by CRP of reactions of opsonic adherence and phagocytosis as outlined in these studies points to a significant role for CRP in reactions of host defense and inflammation.     

Phagocytosis of Plasmodium chabaudi: modulation by immune serum and antigen in vitro

The phagocytosis of free Plasmodium chabaudi parasite by resident peritoneal macrophages of mouse was studied in an in vitro system. The effect of antimalarial antiserum (HIS) was assessed by preincubation of parasite macrophages and both parasite and macrophages with HIS prior to use in phagocytic assays. Highest phagocytic index was obtained with HIS pretreated parasites. The two activities viz. opsonic (parasite dependent) and cytophilic (macrophage dependent) were noted to operate independent of each other. The phagocytosis promoting activity was found to be complement dependent. The receptor site for binding of HIS opsonized but not medium opsonized parasite on the surface of macrophages was blocked by pretreatment of these cells with HIS-soluble antigen combination.     

Role of serum factors in the phagocytosis of weakly or heavily encapsulated Cryptococcus neoformans strains by guinea pig peripheral blood leukocytes

We investigated the opsonic activity of the serum factors affecting phagocytosis of Cryptococcus neoformans in vitro to elucidate the role of humoral factors in the host defense mechanisms against cryptococcosis. Two strains of C. neoformans, one heavily and one weakly encapsulated, were used. Guinea pig peripheral blood leukocytes (PBLs) were used for phagocytosis. The viable weakly encapsulated cells were ingested effectively by PBLs, in the presence of guinea pig normal fresh serum, while the heavily encapsulated cells were not ingested. Neither immune serum, its IgG fraction alone, nor heated serum promoted the phagocytosis of either the weakly or heavily encapsulated strain. On the other hand, immune serum promoted adherence of PBLs to viable cells of the heavily encapsulated strain, forming rosettes in the presence of fresh serum. A substantial amount of C3b component was detected on yeast cells when weakly encapsulated cells were incubated with human fresh serum, or heavily encapsulated cells were incubated with rabbit immune serum together with human fresh serum. Serum chelation experiments also indicated that the factors involved in the alternative complement pathway are opsonins for the weakly encapsulated strain. These results suggest that the alternative pathway plays an important normal opsonic role for weakly encapsulated strains and that specific antibody plays an immune opsonic role for heavily encapsulated strains of C. neoformans via the classical pathway of complement activation.     

Opsonic activity of guinea pig serum is reduced by in vivo administration of carcinogenic nitrosamines

The administration of each of four carcinogenic nitrosamines to normal guinea pigs resulted in decreased opsonic activity of their sera but did not affect the capacity of their peritoneal macrophages to phagocytize particles opsonized with normal serum. Diphenylnitrosamine, a noncarcinogenic analogue, had no significant effect on opsonic or phagocytic activity.     

Importance of complement 3 and mannose receptors in phagocytosis of Paracoccidioides brasiliensis conidia by Nramp1 congenic macrophages lines

Genetic factors influence susceptibility to Paracoccidioidomycosis, a Latin American endemic mycosis. The pattern of susceptibility of congenic mouse strains infected with Paracoccidioides brasiliensis resembles the pattern of the Nramp1 gene. Thus, congenic murine bone-marrow-derived macrophage lines B10R (Nramp1rGly169) and B10S (null Nramp1 protein expression, Nramp1sAsp169) were infected with P. brasiliensis conidia and compared, under opsonic and nonopsonic conditions. Opsonization increased the percentage of phagocytosis by both cell lines. B10R macrophages exhibited a higher percentage of cells with associated conidia and higher number of conidia per macrophage than B10S. Heat-inactivation and EDTA treatment of serum used for opsonization, and treatment of macrophages with anti-complement receptor 3 (CR3) decreased phagocytosis by both cell lines. alpha-methyl-d-mannoside reduced phagocytosis by B10R macrophages, suggesting that the mannose receptor participates in phagocytosis by these cells. The CR3 expression was similar on both cell lines and B10R expressed more mannose receptors, but neither cell line expressed CR1. IFNgamma decreased the conversion of conidia to the yeast form of P. brasiliensis in B10R, but not in B10S macrophages.     

Plasmodium falciparum: identification and purification of the phosphoglycerate kinase of the malaria parasite.

Multiplication of the human malaria parasite Plasmodium falciparum within red blood cells is an energy-dependent process and glucose consumption increases dramatically in infected red blood cells (IRBC) versus normal red blood cells (NRBC). The major pathway for glucose metabolism in P. falciparum IRBC is anaerobic glycolysis. Phosphoglycerate kinase (PGK) is one of the key enzymes of this pathway as it generates ATP. We found that the PGK specific activity in P. falciparum IRBC is seven times higher than that in NRBC. The parasitic origin of the increase in PGK activity is confirmed by isoelectric focusing. Indeed, two P. falciparum isoenzymes with neutral isoelectric points were detected. P. falciparum PGK in purified form has a molecular mass of 48 kDa. Antiserum raised against purified P. falciparum PGK specifically recognizes the 48-kDa protein band in P. falciparum and also reacts with P. berghei and P. yoelii IRBC lysates but does not cross-react with PGK associated with NRBC.     

Migration inhibition of alloimmune mouse macrophages by soluble transplantation antigens

Specific allogeneic transplantation antigens were solubilized and shown to inhibit the migration of alloimmune macrophages. Alloimmune macrophages treated with trypsin prior to antigen exposure migrated in the presence of the specific soluble antigens. The arming of nonimmune macrophages and the rearming of trypsinized immune macrophages with hyperimmune serum was readily observable using the migration inhibition test, whereas immune serum was ineffective.     

Use of hyperimmune serum in treatment of endometritis in mares

Endometritis was induced in four progesterone-treated ovariectomized mares and in two seasonally anestrous mares by intrauterine infusion of Streptococcus zooepidemicus. The bacteria were suspended in phosphate buffered saline (PBS), or in nonimmune or hyperimmune serum. Uterine lavage was performed after 24 h. Significantly fewer (P < 0.05) bacteria were recovered from mares which received hyperimmune serum than from mares which received nonimmune serum or PBS. There was no significant difference between treatment with nonimmune serum or PBS. It is therefore suggested that increasing the availability of specific antibody reduced the severity of intrauterine infection.     

Hormonal and immunological changes in blood and mammary secretion in the sow at parturition

The purpose of the present study was to record possible variations of estradiol-17 beta (E2) and cortisol concentrations, and parameters related to granulocyte phagocytosis in mammary secretions from healthy sows at parturition. The study was comprised 8 primiparous sows (Landrace x Yorkshire). Blood and mammary secretion samples were collected twice daily from 3 d before (only blood) until 3 d after farrowing. Estradiol-17 beta and cortisol concentrations were determined in plasma and in cell-depleted skimmed mammary secretions. Phagocytic capacity of polymorphonuclear cells (PMN) was assessed in whole blood and in cell suspensions derived from mammary secretions. Opsonic activity was assessed in serum and in cell-depleted skimmed mammary secretions. The 2 assays were based on chemiluminescence. Estradiol-17 beta concentration in plasma decreased (P < 0.001) directly after parturition. In skimmed secretions, the highest E2 concentration was recorded in the first sample after parturition and decreased (P < 0.01) thereafter. The highest cortisol concentration in plasma was recorded in the evening before parturition (P < 0.01). In skimmed secretions, there was no significant variation in cortisol concentration. The concentrations of both steroid hormones were lower in mammary secretions than in plasma. The phagocytic capacity of PMN in blood and mammary secretion, expressed as peak chemiluminescence per PMN, showed no significant change. This was also true for the opsonic activity in serum. In skimmed secretions the opsonic activity increased (P < 0.01) after parturition. These data emphasize the differences between plasma and mammary secretion concentrations of steroid hormones as well as between systemic and mammary gland immune competence. Regarding the phagocytosis process in mammary secretions, the part directly related to the PMN function seemed not to be altered at parturition compared with later on in lactation, whereas the part related to opsonic activity seemed to be impaired at parturition. The latter may play a role in the development of coliform mastitis at this time.     

Chromium(III) Nanoparticles Affect Hormone and Immune Responses in Heat-Stressed Rats

This study was conducted to evaluate the effects of chromium nanoparticles (CrNano) on the hormone and immune responses of rats in heat stress condition. A total of 80 male Sprague–Dawley rats were randomly assigned to four dietary treatment groups (n?=?20). The first group was offered a basal diet as a control. The second, third, and fourth groups received basal diet supplemented with 150, 300, and 450 μg/kg Cr, respectively, in the form of CrNano. At the end of the 8-week trial, growth performance, food utilization, and sera concentrations of hormones, immunoglobulins, and alexins were determined. Lymphocyte proliferation activity, antibody response to injected sheep red blood cells (SRBCs), and phagocytosis of peritoneal macrophages were determined by ³H-thymidine uptake method, plaque-forming cells (PFC) assay, and ingesting chicken red blood cells test, respectively. The results indicated that rats that received CrNano exhibited no changes in growth rate and food efficiency compared to the control group. However, dietary supplementation of 150, 300, and 450 μg/kg Cr from CrNano significantly decreased serum concentrations of insulin and cortisol, increased sera levels of insulin-like growth factor I and immunoglobulin G, and enhanced the lymphoproliferative response, anti-SRBC PFC response, and phagocytic activity of peritoneal macrophages. These results suggest that dietary supplementation of Cr as CrNano affects hormone and immune status in heat-stressed rats.     

Opsonizing activity of the serum of gnotobiotic guinea pigs contaminated with individual representatives of intestinal microflora]

The influence of contamination of germfree guinea pigs with individual representatives of the intestinal microflora (Bac. mesentericus, Bac. subtilis, S. albus, and S. faecalis) on the formation of the serum opsonic activity was studied. An increase of the opsonic activity to all the microorganisms on the 11th day after a corresponding monocontamination and a stimulating influence of the serum on the intracellular digestion of Bac. mesentericus and Bac. subtilis microbes was noted. As to the pathogenic microorganisms (E. coli 055), S. Faecalis only were capable of stimulating the serum opsonic activity. The results indicated the presence of an association between the microflora composition and the opsonic activity of the animal blood serum. The value of this index also depended on the properties of the phagocytosis object.     

应用双光子显微镜和流式细胞仪定性及定量确定小鼠巨噬细胞的吞噬功能

建立了流式细胞仪和双光子激光共聚焦荧光显微镜进行定性和定量检测小鼠巨噬细胞吞噬鸡红细胞的方法,并同传统光学显微镜细胞化学染色观察方法相比较,探讨其检测巨噬细胞吞噬效应的优越性。常规方法获取小鼠腹腔和脾脏巨噬细胞,制备巨噬细胞悬液。常规制备鸡红细胞,计数并调整活细胞数,用5-二醋酸羧基荧光素琥珀酸单胞菌酯(5-carboxyfluorescein diacetate succinimidyl ester,CFSE)染色,与巨噬细胞共温育一定时间后,小鼠巨噬细胞特异性荧光抗体F4/80标记巨噬细胞。应用流式细胞仪检测巨噬细胞中CFSE阳性百分率来表示巨噬细胞吞噬率;应用双光子显微镜观察被吞噬的CFSE阳性鸡红细胞动态分布情况。同时,采用传统光学显微镜吉姆萨染色观察巨噬细胞吞噬百分率。结果显示,流式细胞仪结合双光子显微镜检测巨噬细胞吞噬率与传统的显微镜计数法比较,两者有明显的正相关性。双光子显微镜和流式细胞仪可以定性与定量检测巨噬细胞吞噬功能,该方法具有灵敏、快捷、重复性好以及准确率高的特点,是进行免疫学研究的可行方法。     

Mouse Virulence of K(L) Antigen-Containing Strains of Escherichia coli

The mouse virulence of two K antigen-containing (L variety) strains of Escherichia coli (serotype O2:K1) isolated from human septicemia, and of their variants which lacked K antigen, was studied. The strains containing envelope antigen (K+) were highly virulent when injected intracerebrally or when suspended in mucin and injected intraperitoneally. After intraperitoneal injection of E-107 K+ (but not K−), there was a marked initial growth in the peritoneal cavity followed by bacteremia and infection of all the organs examined. In the mucin-enhanced lethal infection, this growth continued until death of the animal; in the nonlethal infection, growth ceased and the count dropped quickly after approximately 5 hr. Host defenses were depressed greatly by intraperitoneally, but not intravenously, administered mucin. Bacteria were most virulent when injected intraperitoneally. In vitro phagocytosis of the K+ bacteria required opsonins not needed for phagocytosis of the smooth K− variants. Opsonins were found in immunized rabbit and normal mouse sera. Immune rabbit sera contained antibodies with anti-K specificity which were opsonic in vitro and highly protective in vivo when administered passively. There appears to be a lesser anti-O opsonic and protective activity involving one of the strains (E-107 K+), and colonial morphology, agglutination, and absorption tests indicated a low amount of K antigen on this organism. No anti-O opsonic or protective activity could be shown involving the other strain (E-102 K+). When standard serological typing procedures were used, these two strains appeared to be identical serologically, but they differed greatly in sensitivity to immune rabbit serum in phagocytosis experiments in vitro.