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Pentraxin 3 (PTX3) is a tumor necrosis factor and interleukin-1beta-stimulated gene that encodes a long PTX with proinflammatory activity. Here, we show that peritoneal macrophages derived from PTX3 transgenic (Tg) mice express higher levels of PTX3 mRNA than macrophages from wild-type (WT) mice, at basal level as well as upon stimulation with zymosan (Zy). Macrophages from Tg mice also showed improved opsonin-independent phagocytosis of Zy particles and the yeast form of the fungus Paracoccidioides brasiliensis. In the case of P. brasiliensis, an enhanced microbicidal activity accompanied by higher production of nitric oxide was also observed in macrophages from Tg mice. Using fluorescein-activated cell sorter analysis and reverse transcriptase-polymerase chain reaction, we demonstrated that basal level of Toll-like receptor-6 and Zy-induced dectin-1 expression was slightly but consistently higher in macrophages from Tg mice than in macrophages from WT mice. Recombinant (r)PTX3 protein binds to Zy particles as well as to yeast cells of P. brasiliensis and addition of rPTX3, to a culture of WT-derived macrophages containing Zy leads to an increase in the phagocytic index, which parallels that of Tg-derived macrophages, demonstrating the opsonin-like activity of PTX3. It is important that blockade of dectin-1 receptor inhibited the phagocytosis of Zy particles by WT and PTX3 Tg macrophages, pointing out the relevant role of dectin-1 as the main receptor involved in Zy uptake. Our results provide evidence for a role of PTX3 as an important component of the innate-immune response and as part of the host mechanisms that control fungal recognition and phagocytosis.

Phagocytes play a central role in the host defense system, and the relationship between the mechanism of their activation and cytoskeletal reorganization has been studied. We have previously reported a possible involvement of cofilin, an actin-binding protein, in phagocyte functions through its phosphorylation/dephosphorylation and translocation to the plasma membrane regions. In this work, we have obtained a new line of evidence showing an important role of cofilin in phagocyte functions using the mouse macrophage cell line J774.1 and an antisense oligonucleotide to cofilin. Upon stimulation with opsonized zymosan (OZ), cofilin was phosphorylated, and it accumulated around phagocytic vesicles. As the antisense oligonucleotide to cofilin, a 20-mer S-oligo corresponding to the sequence including the AUG translational initiation site was found to be effective. In the cells treated with the antisense oligonucleotide, the amount of cofilin was less than 30% of that in the control cells, and the level of F-actin was two or three times higher than that in the control cells before and throughout the cell activation. In the antisense oligonucleotide-treated cells, OZ-triggered superoxide production was three times faster than that in the control cells. Furthermore, phagocytosis of OZ was enhanced by the antisense. These results show that cofilin plays an essential role in the control of phagocyte function through regulation of actin filament dynamics.

Serum amyloid P component (SAP) is a member of the pentraxin family of proteins. These proteins are characterized by cyclic pentameric structure, calcium-dependent ligand binding, and frequent regulation as acute-phase serum proteins. SAP is the serum precursor of the P component of amyloid. It binds to a broad group of molecules, including autoantigens, through a pattern recognition binding site. The related pentraxin, C-reactive protein (CRP), is a strong acute-phase reactant in man and an opsonin. We previously determined that the binding of CRP to leukocytes occurs through Fc receptors for IgG (FcgammaR). We now report that SAP also binds to FcgammaR and opsonizes particles for phagocytosis by human polymorphonuclear leukocytes (PMN). Specific, saturable binding of SAP to FcgammaRI, FcgammaRIIa, and FcgammaRIIIb expressed on transfected COS cells was detected using SAP-biotin and PE-streptavidin. Zymosan was used to test the functional consequences of SAP and CRP binding to FcgammaR. Both SAP and CRP bound to zymosan and enhanced its uptake by PMN. This enhanced phagocytosis was abrogated by treatment of PMN with wortmannin, a phosphatidylinositol-3 kinase inhibitor, or with piceatannol, a Syk inhibitor, consistent with uptake through FcgammaR. Treatment of PMN with phosphatidylinositol-specific phospholipase C to remove FcgammaRIIIb also decreased phagocytosis of SAP-opsonized zymosan, but not CRP-opsonized zymosan. These results suggest that SAP may function in host defense. In addition, as SAP binds to chromatin, a major immunogen in systemic lupus erythematosus, it may provide a clearance mechanism for this Ag through FcgammaR bearing cells.

Complement receptor type 3 (CR3) was initially described as an opsonic receptor. Subsequently, CR3-mediated lectin-sugar recognition mechanisms have been shown to play a major role in the nonopsonic phagocytosis of several pathogens, among them Mycobacterium tuberculosis. Little is known about the binding and signal transduction mechanisms operating during nonopsonic ingestion through CR3 of different microorganisms. In the present study, we used CHO cells stably transfected with CR3 to show that CR3 was able to mediate internalization of zymosan and pathogenic mycobacteria (Mycobacterium kansasii and Mycobacterium avium) but not that of nonpathogenic species (Mycobacterium smegmatis and Mycobacterium phlei). A combination of mannan and beta-glucan inhibited the phagocytosis of zymosan but had no effect on M. kansasii ingestion. Among six monoclonal antibodies (MAbs) directed against the CD11b subunit of CR3 that decreased zymosan ingestion, only three inhibited M. kansasii phagocytosis. In particular, MAbs known to block the CR3 lectin site affected only internalization of zymosan. Using U937 macrophages, we observed that zymosan ingestion through CR3 induced superoxide production measured by cytochrome c reduction and by translocation of the NADPH oxidase cytosolic component p47phox to the phagosomal membrane, whereas phagocytosis of viable or heat-killed M. kansasii did not. Furthermore, lack of superoxide anion production during phagocytosis of M. kansasii was not due to inhibition of NADPH oxidase per se or superoxide anion scavenging. Together, our results indicate that (i) nonopsonic phagocytosis of zymosan and M. kansasii by CR3 implicates different molecular mechanisms involving multiple and distinct epitopes of CD11b and (ii) CR3 may transduce different cellular responses depending on the sites mediating nonopsonic phagocytosis.

The luminol-amplified chemiluminescence (CL) activity of murine splenocytes and bone marrow cells was investigated and compared. Phagocytosis was activated by adding starch grains, opsonized zymosan particles (OZP), or phorbol myristate acetate (PMA) to cell suspensions. CL was studied within a concentration range of 3.13 x 10(6)-1.25 x 10(8) splenocytes/ml, and 1.56 x 10(6)-6.25 x 10(7) bone marrow cells/ml. The CL reaction intensity was increasing with rising cell concentration throughout the whole range studied. Starch grains and OZP elicited a unimodal kinetics of the CL response with a single peak after 10 min for splenocytes and 13-16 min for bone marrow cells. PMA activation induced a bimodal reaction with the first peak appearing after 3 min for either of the cell populations, and the second maximal peak being recorded after 6-7 min for splenocytes and 9-10 min for bone marrow cells.

The U937 cell line has been shown to generate tumour necrosis factor alpha (TNF-alpha) in response to soluble stimuli such as PMA and LPS, but only after treatment with GM-CSF. We report here the generation of TNF-alpha from U937 cells following phagocytosis of opsonised zymozan particles without the need for pre-treatment with GM-CSF. The release of TNF-alpha from U937 cells was demonstrated by a specific radioimmunoassay, L929 cell killing and neutrophil 'priming'. The biological activities in the cell supernatant were inhibited by TNF-alpha antiserum. Phagocytosis was required for TNF-alpha production. Non-opsonised zymozan or latex particles which were not phagocytosed or pretreatment with cytochalasin B, which inhibited phagocytosis of opsonised zymozan particles, all failed to trigger TNF-alpha production. Phagocytosis failed to trigger detectable IL-1 generation, and production of IL-6 was insufficient to produce biological effects on neutrophils. The U937 supernatant thus provides a source of human TNF-alpha which can be generated conveniently and cheaply for experimental investigations.

When sera diluted to 5% in a buffer containing calcium and magnesium were incubated with mannan-coated ELISA plates, C4 fragments, properdin and factor B were bound to the plates as well as the expected opsonic C3 fragments, C3b and C3bi. The calcium-dependent lectin mannan-binding protein, which is structurally similar to C1q, was also shown to bind in this assay and analysis of sera from 179 healthy blood donors revealed that the binding levels of all these proteins were highly significantly correlated. Results obtained with a previously described C3b opsonic assay using zymosan also correlated with the mannan-binding levels. When the sera were diluted to 5% in the presence of Mg-EGTA there was no detectable binding of complement proteins to the mannan surface, confirming that no alternative pathway activation occurred at this serum concentration. When sera were diluted to 5% in a buffer containing EDTA in order to study immunoglobulin binding in the absence of complement activation, the levels of bound IgG1, IgG2, IgG3, IgA and IgM antibodies were found to be completely unrelated to the C3bi binding levels previously observed. The results suggest that in this experimental system using low concentrations of serum, mannan-binding protein initiates an antibody-independent mechanism of cleavage of the classical pathway component C4, which subsequently regulates the degree of cleavage of C3 and recruitment of alternative pathway proteins.

Nonopsonized Brucella abortus and bacteria treated with fresh antiserum, heat-inactivated antiserum, or normal bovine serum were evaluated for their ability to stimulate production of superoxide anion and myeloperoxidase-mediated iodination by neutrophils from cattle. Brucella abortus opsonized with fresh antiserum or heat-inactivated antiserum stimulated production of approximately 3 nmol of O2-/10(6) neutrophils/30 min. Similarly treated bacteria also stimulated the binding of approximately 4.3 nmol of NaI/10(7) neutrophils/h to protein. Significant (P less than 0.05) production of O2- and iodination activity by neutrophils were not stimulated by nonopsonized bacteria or organisms treated with normal bovine serum. Seemingly, B abortus stimulated oxidative metabolism in bovine neutrophils; however, the stimulation was dependent on the presence of bacterial associated opsonins.

Human leukocyte phagocytosis of S. aureus and zymosan particles was measured by flow cytometry (FCM). Killing of bacteria was measured by a standard microbiological method. When pooled human serum was heated to 56 degrees C for 30 min, the percentage of phagocytosing polymorphonuclear neutrophilic leukocytes (PMNLs), the rate of phagocytosis and killing of S. aureus by the whole leukocyte population were reduced to about 50% of the control values. The results indicate that the impaired phagocytosis and killing were due to the lack of attachment of bacteria opsonized with heat-stabile serum opsonins, mainly IgG, to 50% of the PMNLs. A prey-predator model was used to compare phagocytosis of S. aureus in heated and control serum. The rate of phagocytosis by those PMNLs that were active in heated serum was the same as that of all the leukocytes in control serum, suggesting that heat-labile serum opsonins did not affect the rate of phagocytosis by these PMNLs. PMNLs and monocytes phagocytosed zymosan particles in control serum, but in heated serum only a fraction of the leukocytes, corresponding to the fraction of monocytes phagocytosed. Thus, all PMNLs seem to be capable of phagocytosis by heat-labile serum opsonins. The combined use of S.aureus and zymosan particles may be of advantage in rapid screening of serum opsonin activities and phagocyte function in infectious and haematological disorders.

C3c fragments released by the actin of trypsin from C3b molecules bound to zymosan may be readily quantitated by conventional gel techniques to give a measurement of the efficiency of C3b opsonization. Using this approach, the rate of deposition of C3b molecules was found to be very rapid in sera known to opsonize yeast normally. In contrast, sera defective in yeast opsonization deposited C3b much more slowly. The C3b elution technique was found to correlate well with both a direct phagocytosis assay using baker's yeast (r = 0.87, P less than 0.001) and a recently described neutrophil iodide uptake assay (r = 0.88, P less than 0.001).

After opsonization with whole human serum, with IgG antibody alone or with C3 fragments alone, the ingestion of zymosan particles by human neutrophils was found to correlate most closely with the efficiency of C3 fragment deposition on the zymosan surface. Selective opsonization of zymosan particles with IgG did not promote phagocytosis at all in suspension, but could promote ingestion after particle-cell contact was induced by centrifugation. In contrast, zymosan particles in suspension selectively opsonized by C3 fragments were ingested as efficiently as those opsonized with whole serum, and we suggest that C3 fragments provide the principal stimulus for phagocytosis of zymosan. The process of vacuolar sealing was not dependent on the state of opsonization of the particles and was significantly more efficient with unopsonized particles, indicating that formation of unsealed vacuoles is not due to a failure of the 'zipper' mechanism of sequential interaction of cell surface receptors and opsonic ligands.

Zymosan opsonisation was determined in sera of 38 normal individuals and 20 children with acute lymphocytic leukemia (ALL). All patients underwent chemotherapy according to the CoALL 82 protocol. Intravenous gammaglobulin (ivGG) was given prophylactically to replace deficient specific antibodies. Zymosan opsonisation in normal sera ranged from 65% to 133% of a serum pool, whereas sera of children with ALL exhibited markedly decreased opsonisation ranging from 7% to 141% (of the pooled serum standard) at different times during an observation period of 20 months. No significant changes could be observed over time, neither induced by the ivGG infusion itself (short term effect) nor during the 20 months observation period (long term effect). Before ivGG therapy was initiated, a positive correlation was found between zymosan opsonisation and complement parameters (CH 50: p less than 0.01; AP 50; p less than 0.001; C3: p less than 0.05). No correlation could be noted between zymosan opsonisation and IgG concentration. Experiments with complement deficient sera clearly demonstrated the dependence of zymosan opsonisation from complement function. In contrast, sera with little or no IgG but intact complement, showed normal zymosan opsonisation. Deficient zymosan opsonisation might contribute to the immune deficiency of ALL patients. The present study suggests, that the zymosan opsonisation cannot be corrected by ivGG infusions.

Human monocyte-derived macrophages phagocytosed zymosan in the absence of serum opsonins. The capacity to ingest zymosan developed after the cells were cultured in vitro for 3 days and was inhibited completely by mannan. We conclude that human monocyte-derived macrophages phagocytose unopsonized zymosan predominantly via mannose receptors.

Serum opsonic activity against zymosan was found to be decreased more markedly in highly diluted O type serum than in the other 3 types, among which no difference was noted. Heat-killed bacteria were equally well opsonized with any type of serum. This peculiar activity of O type serum against zymosan was not affected by the removal of isohemagglutinins. No inhibitory activity was demonstrable in O type serum.

Several factors of immune response are affected by anaesthesia and surgery. Opsonization as a phase of the phagocytic process was studied in ten patients undergoing cholecystectomy under balanced anaesthesia with thiopentone, suxamethonium, pancuronium, N2O + O2, fentanyl, dehydrobenzperidol and enflurane. The luminol-dependent chemiluminescence responses were depressed at the end of surgery in phagocytosis of patient-serum-opsonized zymosan and Bordetella pertussis (P less than 0.05). The responses to Bordetella pertussis were already depressed after the period of presurgery anaesthesia (P less than 0.05). The responses returned to preinduction values by the third postoperative day. Since the decreases could only be observed with diluted serum and there were no infectious complications in the patients, the serum opsonic capacity was considered clinically sufficient during and after anaesthesia and surgery.