The Phagocytic Code Regulating Phagocytosis of Mammalian Cells.

Mammalian phagocytes can phagocytose (i.e. eat) other mammalian cells in the body if they display certain signals, and this phagocytosis plays fundamental roles in development, cell turnover, tissue homeostasis and disease prevention. To phagocytose the correct cells, phagocytes must discriminate which cells to eat using a 'phagocytic code' - a set of over 50 known phagocytic signals determining whether a cell is eaten or not - comprising find-me signals, eat-me signals, don't-eat-me signals and opsonins. Most opsonins require binding to eat-me signals - for example, the opsonins galectin-3, calreticulin and C1q bind asialoglycan eat-me signals on target cells - to induce phagocytosis. Some proteins act as 'self-opsonins', while others are 'negative opsonins' or 'phagocyte suppressants', inhibiting phagocytosis. We review known phagocytic signals here, both established and novel, and how they integrate to regulate phagocytosis of several mammalian targets - including excess cells in development, senescent and aged cells, infected cells, cancer cells, dead or dying cells, cell debris and neuronal synapses. Understanding the phagocytic code, and how it goes wrong, may enable novel therapies for multiple pathologies with too much or too little phagocytosis, such as: infectious disease, cancer, neurodegeneration, psychiatric disease, cardiovascular disease, ageing and auto-immune disease.

Anti-Cryptococcal activity of a furanone derivative-antibiofilm and opsonophagocytic potential.

Cryptococcus neoformans, an encapsulated fungal pathogen is evolving as a major threat to immune-compromised patients and rarely to healthy individuals also. The cell wall bound capsular polysaccharide, melanin pigment and biofilm formation are major virulence factors that are known to contribute to cryptococcal meningitis. In the present study, a furanone derivative, (E)-5-benzylidenedihydrofuran-2(3H)-one (compound-6) was evaluated against biofilm of seven different strains of C. neoformans in melanized and non-melanized condition. In addition, the efficacy of compound-6 in activation of TLR-2, opsonophagocytosis, and modulation of cytokine expression during phagocytosis were studied. During the biofilm study, we found that moderate capsule size favored biofilm formation. Interestingly, the minimum biofilm eradication concentration (MBEC0.5) of melanized biofilm was found to be achieved at 1- to 1.7-fold higher MBEC0.5 of non-melanized cells. The maximum eradication of 77% and 69% of non-melanized and melanized biofilm were observed. The capsule size was reduced to half of its size with marked changes in morphology. Furthermore, expression of TLR2, iNOS and pro-inflammatory cytokines such as TNF-α, IL-12, and IFN-γ were also facilitated by compound-6. The correlation analysis showed a positive correlation between phagocytosis and the expression of TLR-2, iNOS, IL-6, IL-12. Collectively, the significant effect of compound-6, anti-melanization activity, antibiofilmand effective immunomodulant could be an interesting dual strategy drug agonist against cryptococcal meningitis.

Phagocytosis imprints heterogeneity in tissue-resident macrophages.

Tissue-resident macrophages display varying phenotypic and functional properties that are largely specified by their local environment. One of these functions, phagocytosis, mediates the natural disposal of billions of cells, but its mechanisms and consequences within living tissues are poorly defined. Using a parabiosis-based strategy, we identified and isolated macrophages from multiple tissues as they phagocytosed blood-borne cellular material. Phagocytosis was circadianally regulated and mediated by distinct repertoires of receptors, opsonins, and transcription factors in macrophages from each tissue. Although the tissue of residence defined the core signature of macrophages, phagocytosis imprinted a distinct antiinflammatory profile. Phagocytic macrophages expressed CD206, displayed blunted expression of Il1b, and supported tissue homeostasis. Thus, phagocytosis is a source of macrophage heterogeneity that acts together with tissue-derived factors to preserve homeostasis.

Lipopolysaccharide-mediated enhancement of zymosan phagocytosis by RAW 264.7 macrophages is independent of opsonins, laminarin, mannan, and complement receptor 3.

BACKGROUND: Fungal and bacterial coinfections are common in surgical settings; however, little is known about the effects of polymicrobial interactions on the cellular mechanisms involved in innate immune recognition and phagocytosis. MATERIALS AND METHODS: Zymosan particles, cell wall derivatives of the yeast Saccharomyces cerevisiae, are used to model fungal interactions with host immune cells since they display carbohydrates, including beta-glucan, that are characteristic of fungal pathogens. Using in vitro cell culture, RAW 264.7 macrophages were challenged with zymosan, and phagocytosis determined via light microscopy. The effects of different concentrations of lipopolysaccharide (LPS) on zymosan phagocytosis were assessed. In addition, the transfer of supernatant from LPS-treated cells to naïve cells, the effects of soluble carbohydrates laminarin, mannan, or galactomannan, and the impact of complement receptor 3 (CR3) inhibition on phagocytosis were also determined. RESULTS: LPS enhanced phagocytosis of zymosan in a dose-dependent manner. Transfer of supernatants from LPS-primed cells to naïve cells had no effect on phagocytosis. Laminarin inhibited zymosan phagocytosis in naïve cells but not in LPS-primed cells. Neither mannan, galactomannan, nor CR3 inhibition had a significant effect on ingestion of unopsonized zymosan in naïve or LPS-treated cells. CONCLUSIONS: Zymosan recognition by naïve cells is inhibited by laminarin, but not mannan, galactomannan, or CR3 inhibition. LPS enhancement of phagocytosis is laminarin insensitive and not mediated by supernatant factors or zymosan engagement by the mannose or CR3 receptors. Our data suggest alternative mechanisms of zymosan recognition in the presence and absence of LPS.

Recombinant form of human wild type mannan-binding lectin (MBL/A) but not its structural variant (MBL/C) promotes phagocytosis of zymosan by activating complement.

Mannan-binding lectin (MBL) mediates innate immune responses, such as activation of the complement lectin pathway and phagocytosis, to help fight infections. In the present study, employing recombinant forms of human MBL (rMBL), the role of wild type MBL (rMBL/A) and its structural variant rMBL/C in mediating THP-1 phagocytosis of fluorescent-labeled zymosan was examined and compared to MBL purified from human plasma (pMBL/A). Flow cytometric analyses revealed that opsonization of zymosan with rMBL/A and pMBL/A (0.5-30microg/ml) resulted in a 1.9- and 2.7-fold enhancement in its uptake by THP-1 cells in the presence of serum that was depleted of both MBL and the classical pathway component, C1q (MBL/C1q Dpl serum). In contrast, no enhancement in phagocytosis was observed when zymosan was opsonized with rMBL/C. Addition of MBL monoclonal antibody, EDTA, or mannan to the opsonization reaction mixture inhibited THP-1 phagocytosis of pMBL/A opsonized zymosan. Heat inactivation of MBL/C1q Dpl serum abolished the 2-fold increase in phagocytosis and in the absence of serum the direct opsonic activity of MBL did not contribute significantly to the uptake of zymosan into THP-1 cells. Activation products of complement components C3 and C4 were deposited on zymosan opsonized with pMBL/A and rMBL/A but not rMBL/C indicating that MBL-mediated phagocytosis of zymosan requires activation of the complement lectin pathway. The findings imply that impaired MBL-mediated phagocytosis may put individuals homozygous for the mutant allele MBL/C but not wild type MBL/A at increased risk to infections such as yeast.

Mannose-binding lectin (MBL) substitution: recovery of opsonic function in vivo lags behind MBL serum levels.

Mannose-binding lectin (MBL) deficiency is often associated with an increased risk of infection or worse prognosis in immunocompromised patients. MBL substitution in these patients might diminish these risks. We therefore performed an open, uncontrolled safety and pharmacokinetic MBL-substitution study in 12 pediatric oncology patients with chemotherapy-induced neutropenia. Twice weekly MBL infusions with plasma-derived MBL yielded MBL trough levels >1.0 microg/ml. We tested whether MBL substitution in vivo increased MBL-dependent complement activation and opsonophagocytosis of zymosan in vitro. Upon MBL substitution, opsonophagocytosis by control neutrophils increased significantly (p < 0.001) but remained suboptimal, although repeated MBL infusions resulted in improvement over time. The MBL-dependent MBL-associated serine protease (MASP)-mediated complement C3 and C4 activation also showed a suboptimal increase. To explain these results, complement activation was studied in detail. We found that in the presence of normal MASP-2 blood levels, MASP-2 activity (p < 0.0001) was reduced as well as the alternative pathway of complement activation (p < 0.05). This MBL-substitution study demonstrates that plasma-derived MBL infusions increase MBL/MASP-mediated C3 and C4 activation and opsonophagocytosis, but that higher circulating levels of plasma-derived MBL are required to achieve MBL-mediated complement activation comparable to healthy controls. Other patient cohorts should be considered to demonstrate clinical efficacy in phase II/III MBL-substitution studies, because we found a suboptimal recovery of (in vitro) biological activity upon MBL substitution in our neutropenic pediatric oncology cohort.

Osteopontin functions as an opsonin and facilitates phagocytosis by macrophages of hydroxyapatite-coated microspheres: implications for bone wound healing.

Osteopontin (OPN) is a secreted protein abundant in mineralized tissue extracellular matrices and bodily fluids. Previously we have shown that mineralized debris at surgical wound sites in bone and teeth are coated by macrophage-derived OPN and phagocytosed. Here, we have performed opsonophagocytosis assays to determine whether OPN acts as an opsonin and facilitates phagocytosis by macrophages of protein- and hydroxyapatite mineral-coated microspheres. Moreover, we have examined the opsonization effects of monomer OPN versus OPN polymerized (crosslinked) by tissue transglutaminase 2. Murine macrophages J774A.1 were exposed to polystyrene-latex microspheres having different surface chemistries (non-ionic, aldehyde amidine, carboxyl and aliphatic amine) which were coated with either serum albumin, immunoglobulin, monomer OPN or polymer OPN. Similar experiments with the same protein coatings were performed using hydroxyapatite-covered microspheres. Internalization of microspheres by phagocytosis into macrophages was confirmed by co-localization with the (phago)lysosomal markers lysosome-associated membrane protein-1 (Lamp-1) and LysoTracker, and by light microscopy and transmission electron microscopy after serial sectioning of plastic/resin-embedded cells containing microspheres. OPN significantly increased phagocytosis of both microspheres and hydroxyapatite-covered microspheres compared to negative controls (albumin-coated and uncoated microspheres), with phagocytic indices similar to, or greater than, those of the positive control (IgG-coated). The effect of OPN and hydroxyapatite on microsphere phagocytosis was synergistic. Polymer OPN further enhanced the phagocytosis of aliphatic amine and aldehyde amidine microspheres. Taken together, these results indicate that OPN is an effective opsonin able to facilitate particle uptake (including mineralized particles) by macrophages.

Vitellogenin functions as a multivalent pattern recognition receptor with an opsonic activity.

BACKGROUND: Vitellogenin (Vg), a major reproductive protein, has been associated with infection-resistant response in fish. However, the underlying mechanisms by which Vg is involved in anti-infectious response are not understood. METHODOLOGY/RESULTS: By both protein-microbe interaction analysis and enzyme-linked immunosorbent assay as well as phagocytosis test, we demonstrate for the first time that fish Vg acts as a pattern recognition molecule with multiple specificities that can recognize bacteria as well as fungus rather than self components from fish, and functions as an opsonin that can enhance macrophage phagocytosis. CONCLUSIONS: This study shows that fish Vg plays an integrative function in regulating immunity via its pleiotropic effects on both recognizing pathogen-associated molecular patterns and promoting macrophage phagocytosis. It also supports the notion that factors normally involved in control of female reproduction are associated with immunity in organisms that rely on Vg for oocyte development.

Efficient clearance of opsonised apoptotic cells in the absence of PECAM-1.

It is now accepted that the recognition and uptake of apoptotic cells by phagocytes is a complex process involving a large number of opsonins, receptors and ligands, however the relative contribution of all these molecules are still debated. Here we examined the role of CD31 (PECAM-1) in the recognition/uptake of apoptotic thymocytes by murine bone marrow-derived macrophages (BMDM) in vitro, and by resident peritoneal macrophages in vivo. In the absence of serum, CD31 deficiency, on either the phagocyte or the apoptotic cell, resulted in a reduction in the clearance of apoptotic thymocytes, when a high ratio of apoptotic cells to macrophages was applied. In the presence of serum however there was no discernible contribution of CD31 to the clearance of apoptotic cells by bone marrow-derived macrophages, irrespective of the ratio of cells used. In vivo peritoneal clearance experiments confirmed that in the presence of soluble opsonins CD31 deficiency had no effect on this process. These data suggest that the overall role played by CD31 in the ingestion of apoptotic cells is negligible and most likely overwhelmed by the effects of serum opsonins, such as complement components.

Cell-mediated immunity in arthropods: hematopoiesis, coagulation, melanization and opsonization.

The functions of hemocytes in innate immune response are reviewed with emphasized on their roles in coagulation, melanization and opsonization. Also the ways in which hemocytes are produced in and released from hematopoietic tissue are discussed.

Antibody- and complement-independent phagocytotic and cytolytic activities of human macrophages toward porcine cells.

BACKGROUND: It has been speculated that host macrophages contribute to rapid clearance of transplanted xenogeneic cells. To address such a possibility, phagocytotic and cytolytic activities of human macrophages toward xenogeneic porcine cells were evaluated in vitro in the absence of antibodies and complement factors. METHODS: Human peripheral monocyte-derived macrophages (P-macrophages) and reticulo-endothelial macrophages (RE-macrophages) were obtained from volunteers' peripheral blood and from the perfusion effluents of liver allografts for transplantation, respectively. 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled human autologous red blood cells (auto-RBCs), ABO-incompatible RBCs (incom-RBCs) and xenogeneic porcine RBCs (xeno-RBCs) were incubated with the human macrophages; subsequently, the macrophages that had phagocytosed the RBCs could be identified as CFSE positive cells by FCM analyses and confocal microscopy. Cytolytic activity was quantified by calculating levels of lactate dehydrogenase in each culture supernatant. RESULTS: Human RE-macrophages spontaneously phagocytosed and had a remarkable cytolytic activity toward xeno-RBCs, but not toward auto-RBCs or incom-RBCs. Elimination of alpha-galactosyl xenoantigen (alpha-Gal) epitopes on xeno-RBCs did not prevent phagocytotic or cytolytic activity of RE-macrophages. CONCLUSIONS: These findings indicate phagocytotic and cytolitic activities of human macrophages toward porcine cells are initiated by a factor other than alpha-Gal in a mechanism independent of antibody/complement opsonization.

Exoenzyme Tat-C3 inhibits association of zymosan particles, phagocytosis, adhesion, and complement binding in macrophage cells.

Phagocytosis by macrophages is most important in the initial stages of an immune response. Although RhoA regulates cell adhesion, its roles in the integrin-related association of particles with macrophages and in phagocytosis are not clearly understood. We introduced C3 exoenzyme, a specific inhibitor of Rho, into J774A.1 macrophage cells fused with the 9 amino acid (49-57) transduction domain (RKKRRQRRR) of HIV-1 Tat. The presence of this Tat-C3 vector altered RhoA mobility on non-denaturing gels, indicating that Tat-C3 modified RhoA by ADP-ribosylation. Uptake of (FITC)-conjugated serum-opsonized zymosan particles and adhesion to fibrinogen-coated plates were reduced as was the association of serum-opsonized zymosan particles, and complement C3 and C3bi with the transfected cells. These results suggest that Rho regulates the activity of integrins that are involved in the association of particles with macrophages, phagocytosis, adhesion, and binding of complement C3 and C3bi.

Trojan horse effect: phagocyte-mediated Streptococcus iniae infection of fish.

The salmonid macrophage-like cell line RTS-11 and purified trout pronephros phagocytes were used to analyze in vitro entry and survival of two Streptococcus iniae serotypes. Efficient invasion by S. iniae occurred in both cells, but only the type II strain persisted in pronephros phagocytes for at least 48 h. Ex vivo models of opsonin-dependent phagocytosis by pronephros phagocytes demonstrated increased phagocytosis efficacy. Analysis of phagocytes collected from diseased fish demonstrated that approximately 70% of the bacteria contained in the blood during the septic phase of the disease were located within phagocytes, suggesting an in vivo intracellular lifestyle. In addition to the augmented levels of bacteremia and enhanced survival within phagocytes, S. iniae type II induces considerable apoptosis of phagocytes. These variabilities in intramacrophage lifestyle might explain differences in the outcomes of infections caused by different serotypes. The generalized septic disease associated with serotype II strains is linked not only to the ability to enter and multiply within macrophages but also to the ability to cause considerable death of macrophages via apoptotic processes, leading to a highly virulent infection. We assume that the phenomenon of survival within phagocytes coupled to their apoptosis plays a crucial role in S. iniae infection. In addition, it may provide the pathogen an efficient mechanism of translocation into the central nervous system.

Histidine and aspartic acid residues important for immunoglobulin G endopeptidase activity of the group A Streptococcus opsonophagocytosis-inhibiting Mac protein.

The secreted Mac protein made by serotype M1 group A Streptococcus (GAS) (designated Mac(5005)) inhibits opsonophagocytosis and killing of GAS by human polymorphonuclear neutrophils. This protein also has cysteine endopeptidase activity against human immunoglobulin G (IgG). Site-directed mutagenesis was used to identify histidine and aspartic acid residues important for Mac IgG endopeptidase activity. Replacement of His262 with Ala abolished Mac5005 IgG endopeptidase activity. Asp284Ala and Asp286Ala mutant proteins had compromised enzymatic activity, whereas 21 other Asp-to-Ala mutant proteins cleaved human IgG at the apparent wild-type level. The results suggest that His262 is an active-site residue and that Asp284 and Asp286 are important for the enzymatic activity or structure of Mac protein. These Mac mutants provide new information about structure-activity relationships in this protein and will assist study of the mechanism of inhibition of opsonophagocytosis and killing of GAS by Mac.

Localized exocytosis of primary (lysosomal) granules during phagocytosis: role of Ca2+-dependent tyrosine phosphorylation and microtubules.

The uptake and killing of bacteria by human neutrophils are dependent on the fusion of secretory granules with forming phagosomes. The earliest component of exocytosis was found to precede phagosome closure, so that granular membrane constituents were detectable on the plasmalemma. We show that during phagocytosis of IgG-opsonized particles, this early secretory response is highly polarized in the case of primary granules, but less so for specific granules. The vectorial discharge of primary granules was dependent on calcium, but no evidence was found that calcium is involved in determining the polarity of exocytosis. In particular, a redistribution of endomembrane calcium stores toward forming phagosomes could not be detected. Polarized granule exocytosis was accompanied by focal tyrosine phosphorylation and actin polymerization, although the latter was not required for the response. Instead, microtubules seemed to contribute to the vectorial nature of the response. During particle ingestion, the microtubule-organizing center relocated toward forming phagosomes, and colchicine treatment altered the pattern of exocytosis, reducing its directionality. We hypothesize that the focal activation of tyrosine kinases generates localized signals that induce exocytosis in a calcium-dependent manner, and that reorientation of microtubules facilitates preferential delivery of granules toward the forming phagosome.

[Effect of thymic peptides on the functional activity of phagocytic cells of donor peripheral blood]. / Vliianie timicheskikh peptidov na funktsional'nuiu aktivnost' fagotsitarnykh kletok perifericheskoi krovi donorov.

The direct action of synthetic peptide preparations, analogous to thymic hormones, on the functions of phagocytic cells was studied. The preparations Thymogen, Neogen and Thymodepressin in a dose of 10 mM produced a stimulating effect on the ingestive activity of the neutrophil, but not monocytic, population. All three preparations also enhanced the formation of oxygen metabolites registered in the luminol-dependent chemiluminescent analysis. The characteristics of spontaneous chemiluminescence (CL) reflecting the basal level of the synthesis of the active forms of oxygen and CL induced by opsomized zymosan significantly increased also in those cases when the preparations were used in a dose of 10 mM. The level of the synthesis of hydrogen peroxide in individual cells could be appraised by the intensity of the luminescence of dichlorofluorescein diacetate (DCF-DA), evaluated with the use of flow cytometry. All preparations produced a stimulating effect on the formation of hydrogen peroxide in monocytes. The reaction of neutrophils was even more active: Neogen (10 mM) produced the twofold change in the intensity of the luminescence of DCF-DA) in neutrophils, Thymogen and Thynodepressin increased the average intensity of the luminescence of DCF-DA by 80% and 60%, respectively.

Interactions of liposomes with cells in vitro and in vivo: opsonins and receptors.

A number of studies have appeared recently on the underlying mechanisms of liposome-cell interactions under in vitro conditions, in which isolated cell populations or cell lines were used. However, our knowledge of how liposomes interact with cells and the parameters that influence this in vivo is limited. We will summarize and discuss the relevant studies on this matter in this article. In addition, researchers in this field have long been aware of the interaction of liposomes with blood (or serum/plasma) proteins in vivo and their potential role in the process of the clearance of liposomes from the circulation. Some of the 'opsonizing' proteins, such as complement components, immunoglobulins, which enhance the interactions of liposomes with 'phagocytic cells' have been identified. However, the issue of which types of opsonins determine the fate of liposomes in vivo and how liposomal physicochemical properties such as size, charge and fluidity play an important role in the process of liposome clearance is not clear. Our own observations of one of opsonins, complement component are reviewed herein. As opposed to the fate of conventional liposomes, we briefly touch on the interaction of surface-modified liposomes, which are designed to avoid interactions with blood proteins and/or cells (sterically stabilized liposomes, long-circulating liposomes) and to actively target specific cells or tissues (targeted liposomes: immunoliposomes). Blood proteins such as opsonins are not usually thought to play an important role in the clearance of such liposomes.

Opsonization and phagocytosis of Plasmodium falciparum merozoites measured by flow cytometry.

A flow cytometric phagocytosis assay was established to investigate the role of anti-merozoite antibody, complement, and cytokines on the phagocytosis of Plasmodium falciparum merozoites by human neutrophils. This assay involved allowing fluorescein isothiocyanate-labeled merozoites to interact with phagocytes and analysis of the cells on a FACScan with Lysis II software. To differentiate the proportion of neutrophil surface-bound merozoites from the merozoites ingested by neutrophils, the fluorescence of bound merozoites was quenched by adding trypan blue. The data showed that sera from malaria-immune individuals in the Solomon Islands and Papua New Guinea promoted merozoite engulfment by neutrophils. The cytokines tumor necrosis factor alpha, gamma interferon, granulocyte-macrophage colony-stimulating factor, and interleukin-1beta significantly increased the amount and the rate of merozoite phagocytosis by neutrophils. Optimum merozoite phagocytosis occurred when both cytokines and anti-malarial antibody were present.

Protection from lethal gram-positive infection by macrophage scavenger receptor-dependent phagocytosis.

Infections with gram-positive bacteria are a major cause of morbidity and mortality in humans. Opsonin-dependent phagocytosis plays a major role in protection against and recovery from gram-positive infections. Inborn and acquired defects in opsonin generation and/or recognition by phagocytes are associated with an increased susceptibility to bacterial infections. In contrast, the physiological significance of opsonin-independent phagocytosis is unknown. Type I and II class A scavenger receptors (SR-AI/II) recognize a variety of polyanions including bacterial cell wall products such as lipopolysaccharide (LPS) and lipoteichoic acid (LTA), suggesting a role for SR-AI/II in innate immunity to bacterial infections. Here, we show that SR-AI/II-deficient mice (MSR-A(-/-)) are more susceptible to intraperitoneal infection with a prototypic gram-positive pathogen, Staphylococcus aureus, than MSR-A(+/+) control mice. MSR-A(-/-) mice display an impaired ability to clear bacteria from the site of infection despite normal killing of S. aureus by neutrophils and die as a result of disseminated infection. Opsonin-independent phagocytosis of gram-positive bacteria by MSR-A(-/-) macrophages is significantly decreased although their phagocytic machinery is intact. Peritoneal macrophages from control mice phagocytose a variety of gram-positive bacteria in an SR-AI/II-dependent manner. Our findings demonstrate that SR-AI/II mediate opsonin-independent phagocytosis of gram-positive bacteria, and provide the first evidence that opsonin-independent phagocytosis plays a critical role in host defense against bacterial infections in vivo.

Effect of antiflagellar human monoclonal antibody on gut-derived Pseudomonas aeruginosa sepsis in mice.

We evaluated the effect of antiflagellar human monoclonal antibody on gut-derived Pseudomonas aeruginosa sepsis. Mice were given a suspension of P. aeruginosa SP10052 in their drinking water and were simultaneously treated with ampicillin (200 mg/kg of body weight) to disrupt the normal bacterial flora. Cyclophosphamide was then administered to induce leukopenia and translocation of the P. aeruginosa that had colonized the gastrointestinal tract, thereby producing gut-derived generalized sepsis. In this model, intraperitoneal injection of 100 microg of antiflagellar human monoclonal antibody (SC-1225) per mouse for 5 consecutive days significantly (P < 0.01) increased the survival rate compared with that for mice treated with bovine serum albumin (BSA). Treatment with SC-1225 significantly reduced the average number of viable bacteria in portal blood, liver, and heart blood compared with the average number after treatment with BSA. Furthermore, the presence in serum of the inflammatory cytokines tumor necrosis factor alpha and interleukin 6 were evaluated as markers of severity of infection, and the results showed that the levels of these cytokines in mice treated with SC-1225 were significantly decreased in comparison with those in BSA-treated control mice. Although there was no significant difference in the number of bacteria that colonized the intestine, SC-1225 treatment significantly increased bacterial opsonophagocytosis by cultured peritoneal macrophages from mice with or without cyclophosphamide pretreatment. Our results indicate that antiflagellar human monoclonal antibody SC-1225 protects mice against gut-derived sepsis caused by P. aeruginosa and suggest that such an effect is due to its opsonophagocytic activity and the reduced motility of the translocated bacteria once the bacteria move from the intestine into the bloodstream.