Bottom-up, integrated -omics analysis identifies broadly dosage-sensitive genes in breast cancer samples from TCGA.

The massive genomic data from The Cancer Genome Atlas (TCGA), including proteomics data from Clinical Proteomic Tumor Analysis Consortium (CPTAC), provides a unique opportunity to study cancer systematically. While most observations are made from a single type of genomics data, we apply big data analytics and systems biology approaches by simultaneously analyzing DNA amplification, mRNA and protein abundance. Using multiple genomic profiles, we have discovered widespread dosage compensation for the extensive aneuploidy observed in TCGA breast cancer samples. We do identify 11 genes that show strong correlation across all features (DNA/mRNA/protein) analogous to that of the well-known oncogene HER2 (ERBB2). These genes are generally less well-characterized regarding their role in cancer and we advocate their further study. We also discover that shRNA knockdown of these genes has an impact on cancer cell growth, suggesting a vulnerability that could be used for cancer therapy. Our study shows the advantages of systematic big data methodologies and also provides future research directions.

Complement component C1q enhances invasion of human mononuclear phagocytes and fibroblasts by Trypanosoma cruzi trypomastigotes.

Internalization and infectivity of Trypanosoma cruzi trypomastigotes by macrophages is enhanced by prior treatment of parasites with normal human serum. Heating serum or removing C1q from serum abrogates the enhancement, but augmentation of attachment and infectivity is restored by addition of purified C1q to either serum source. Although both noninfective epimastigotes (Epi) and vertebrate-stage tissue culture trypomastigotes (TCT) bind C1q in saturable fashion at 4 degrees C, internalization by monocytes and macrophages of TCT but not Epi-bearing C1q is enhanced in comparison to untreated parasites. Adherence of human monocytes and macrophages to surfaces coated with C1q also induces a marked enhancement of the internalization of native TCT. C1q enhances attachment of both Epi and TCT to human foreskin fibroblasts, but only when C1q is on the parasite and not when the fibroblasts are plated on C1q-coated surfaces. Only TCT coated with C1q show enhanced invasion into fibroblasts. Although trypomastigotes produce an inhibitor of the complement cascade which limits C3 deposition during incubation in normal human serum, C1q binds to the parasite and enhances entry of trypomastigotes into target cells.

Differential expression of endoplasmic reticulum stress-response proteins in different renal tubule subtypes of OVE26 diabetic mice.

Regulation of the endoplasmic reticulum (ER) stress-response pathway during the course of diabetes specifically in renal tubules is unclear. Since tubule cell dysfunction is critical to progression of diabetic nephropathy, this study analyzed markers of ER stress response and ER chaperones at different stages of diabetes and in different renal tubule subtypes of OVE26 type-1 diabetic mice. ER stress-responseinduced chaperones GRP78, GRP94, and protein disulfide isomerase (PDI) were increased in isolated cortical tubules of older diabetic mice, while PDI was decreased in tubules of young diabetic mice. Immunofluorescence staining of kidneys from older mice showed GRP78 and PDI upregulation in all cortical tubule segments, with substantial induction of PDI in distal tubules. Protein kinase RNA-like endoplasmic reticulum kinase (PERK) phosphorylation was increased in cortical tubules of young diabetic mice, with no differences between older diabetic and control mice. Expression of ER stress-induced PERK inhibitor p58IPK was decreased and then increased in all tubule subtypes of young and older mice, respectively. Knockdown of PERK by small interfering RNA (siRNA) increased fibronectin secretion in cultured proximal tubule cells. Tubules of older diabetic mice had significantly more apoptotic cells, and ER stress-induced proapoptotic transcription factor C/EBP homologous protein (CHOP) was increased in proximal and distal tubules of diabetic mice and diabetic humans. CHOP induction in OVE26 mice was not altered by severity of proteinuria. Overexpression of CHOP in cultured proximal tubule cells increased expression of fibronectin. These findings demonstrate differential ER stress-response signaling in tubule subtypes of diabetic mice and implicate a role for PERK and CHOP in tubule cell matrix protein production.

Inactivation of the small GTPase Rho disrupts cellular attachment and induces adhesion-dependent and adhesion-independent apoptosis.

Rho small GTPases regulate a variety of cellular signaling pathways involved in cell growth and transformation. In this study, we examined potential roles for Rho in adhesion-dependent and -independent pathways regulating apoptosis. Rho GTPases are specifically inactivated by exoenzyme C3 (C3) of Clostridium botulinum. Using a novel Sindbis virus-based gene expression system, we created a double subgenomic recombinant (dsSIN:C3) capable of expressing active C3 in intact cells. Infection of L929 fibroblasts with dsSIN:C3 caused essentially complete ADP-ribosylation of intracellular Rho within 1 h. dsSIN:C3-infected cells also became rounded within 1-2 h and detached by 5 h post-infection. Infection of L929 in suspension with dsSIN:C3 disrupted the ability for normal cellular attachment and spreading. Infection of primary cell explants of chicken embryo fibroblasts (CEF) and rat aortic smooth muscle cells (RSM) with dsSIN:C3 caused cytoskeletal effects similar to those seen in L929. We also observed that C3 markedly decreased the basal phosphorylation state of focal adhesion kinase (FAK). Most intriguingly, we found that dsSIN-based expression of C3 or loss of function mutants of Rho could each induce apoptosis and, in RSM, this effect was observed to be adhesion-independent. Rho GTPases, therefore, appear to regulate signal pathways that are required for cell survival and growth that are separate from, but likely overlap with, Rho-dependent pathways involved in cellular adhesion.

A balance of signaling by Rho family small GTPases RhoA, Rac1 and Cdc42 coordinates cytoskeletal morphology but not cell survival.

Rho family GTPases are known to be involved in cytoskeletal reorganization. We examined the possibility that these functions may be dictated by a balance of Rho family GTPase signaling. Using transient viral expression of RhoA, Rac1, Cdc42 and their mutants, as well as C3 exoenzyme, we altered cytoskeletal organization under normal growth conditions. Overexpression of wild-type or constitutively active forms of the Rho family GTPases led to their respective activation phenotypes. Overexpression of dominant negative forms of given Rho family GTPases led to a phenotype consistent with activation of the other Rho family GTPase. Treatment with C. difficile toxin A, that inactivates all Rho family GTPases, led to the transient appearance of a variety of activation phenotypes. Previously, we reported that inactivation of Rho led to induction of apoptosis, implying that Rho may play an important role in cell survival signaling. This signaling, however, is not affected by expression of any forms of Rac1 or Cdc42, and only inactivation of Rho led to induction of apoptosis. Rho family GTPases appear to coordinate cytoskeletal organization by a balance of signaling, while cell survival is regulated by a distinct Rho-mediated signaling pathway.

Clostridium difficile-associated disease in human stem cell transplant recipients: coming epidemic or false alarm?

Clostridium difficile is the most common cause of nosocomial diarrhea in the United States and Europe, and is a cause of significant morbidity and mortality among hospitalized patients. A newly identified epidemic strain has been associated with many hospital outbreaks of C. difficile-associated disease (CDAD), raising the concern of an escalating burden of CDAD among at-risk patients. Hematopoietic SCT (HSCT) recipients are known to be at increased risk for a wide variety of infectious complications, including CDAD as a result of prolonged hospitalizations, exposure to broad-spectrum antibiotics, altered integrity of the intestinal mucosa and GVHD. The incidence of CDAD in the HSCT population has been reported as high as 20% in some large series. The frequency and seriousness of CDAD in this defined group as compared with the general hospital population, however, are not clearly delineated. We discuss the epidemiology and diagnosis of CDAD and review recent studies examining the risk factors and characteristics of CDAD in HSCT recipients. Finally, we provide a management algorithm for the diagnosis and treatment of CDAD at our institution.

Clostridial toxins: molecular probes of Rho-dependent signaling and apoptosis.

The Rho family small GTPases are members of the Ras superfamily of small GTPases. Rho proteins were first determined to act as key regulators of many types of actin cytoskeletal-dependent cellular functions. Recent work by several investigators indicates that Rho GTPases are also critical modulators of several important intracellular and nuclear signal transduction pathways. Certain clostridial toxins and exoenzymes covalently modify, and thereby inactivate, specific types of Rho family GTPases. As such, these microbial enzymes have proven invaluable in helping to identify structural and functional attributes of Rho GTPases.

Human B lymphocyte colony responses. I. General characteristics and modulation by monocytes.

The present study investigated the ability of human B cell-enriched subpopulations to focally proliferate and form colonies in semisolid cultures after stimulation with staph protein A (SpA). After 6 days of incubation, cultures of B-enriched populations exhibited distinct colonies, the number being dependent on the concentration of SpA and the cell density. Optimal colony responses were 1.6 x 10(3) per 1 x 10(6) B lymphocytes, and greater than 83% of the colony-forming cells expressed surface immunoglobulin (sIg). The depletion of adherent monocytes from the B cell-enriched preparations decreased the colony responses approximately 3-fold compared with the nondepleted B cell populations. Adding optimal numbers of adherent monocytes to the monocyte-depleted B cells restored the colony responses; however, less augmentation was observed in single-layer co-cultures containing greater than optimal numbers of monocytes. Identical experiments in double-layer semisolid cultures revealed that relatively greater numbers of monocytes were required to enhance B cell colony responses. Thus, progressively higher ratios of monocytes to B cells resulted in increasing numbers of colonies and failed to demonstrate the diminished colony responses observed in the single-layer system. These studies demonstrate that human B cells form distinct colonies when activated by SpA and that normal adherent monocytes modulate the magnitude of colony responses. Although monocytes predominately enhance B cell clonal differentiation, the evidence presented also suggests that, to a lesser extent, soluble inhibitory materials are elaborated.

Clostridium difficile toxins A and B are cation-dependent UDP-glucose hydrolases with differing catalytic activities.

Toxins A and B of Clostridium difficile are UDP-glucose glucosyltransferases that exert their cellular toxicity primarily through their abilities to monoglucosylate, and thereby inactivate, Rho family small GTPases. Toxin A also hydrolyzes UDP-glucose, although this activity is not well characterized. In this study, we measured the kinetics of UDP-glucose hydrolysis by toxins A and B and found significant differences in the catalytic activities of these two structurally homologous toxins. The toxins displayed similar Michaelis constants (Km) for UDP-glucose, but the maximal velocity (Vmax) of toxin B was approximately 5-fold greater than that of toxin A. Toxins A and B exert their enzymatic actions intracellularly, and, interestingly, we found that each toxin absolutely required K+ for optimal hydrolase activity; Na+ was inactive. The toxins also required certain divalent cations for activity and exhibited a significantly greater Vmax and lower Km in the presence of Mn2+ as compared with Mg2+. We conclude that C. difficile toxins A and B are cation-dependent UDP-glucose hydrolases that differ significantly in their catalytic activities, a finding that may have important implications in understanding their different cytotoxic effects.

Characterization of C1q receptor expression on human phagocytic cells: effects of PDBu and fMLP.

The receptor-mediated binding of C1q to human phagocytic cells was investigated in this study. By using a C1q binding assay, we determined that purified, elutriated monocytes expressed an average of 4.6 X 10(5) C1q receptors (C1qR) per cell, with an equilibrium binding constant (Keq) of 0.91 X 10(7) (M-1). When analyzed in an identical manner, the polymorphonuclear leukocytes (PMN) expressed an average of 4.2 X 10(5) C1qR per cell, with a Keq for C1q of 1 X 10(7) (M-1). Fluorescent flow cytometric analysis showed that C1q was bound by 98% of the monocytes studied. Further, the pattern formed by these cells was consistent with a normal log distribution, indicating that this was a homogeneous population of cells. When PMN were assayed with flow cytometry, however, we found that C1q was bound by an average of only 45% of the PMN analyzed. Further, these PMN were not dispersed in a normal log distribution, indicating some heterogeneity among the cells that bind C1q. We examined the abilities of the chemoattractant N-formylmethionylleucylphenylalanine (fMLP) and the phorbol ester phorbol dibutyrate (PDBu) to modulate expression of C1qR as compared to the receptor for C3b (CR1). Pretreatment of the monocytes and the PMN with either 10(-6)M fMLP or 10 ng/ml of PDBu significantly increased cell surface CR1 expression, as reported previously by other investigators. In contrast, no significant increase in the number of C1qR on the monocytes or the PMN was observed with any of the concentrations of fMLP or PDBu used during pretreatment. However, with certain pretreatment doses of these agents, some reduction was noted in the amount of 125I-C1q bound to the monocytes and the PMN. This study characterizes the binding of C1q to purified monocytes and confirms previously published values for PMN. The distribution of cells expressing C1qR is shown to be significantly different between identically treated populations of monocytes and PMN. Finally, the abilities of fMLP and PDBu to modulate the binding of C1qR are examined. Our results indicate that the control of C1qR expression differs markedly from that of CR1.

C1q enhances the phagocytosis of Cryptococcus neoformans blastospores by human monocytes.

We investigated whether C1q, a subunit of the first component of C, could modulate human peripheral blood monocyte-mediated phagocytosis of Cryptococcus neoformans (CN). Adherence of monocytes to C1q-coated surfaces induced a significant enhancement of ingestion of CN blastospores that had been opsonized with specific anticapsular IgG (IgG-CN). Additionally, C1q enhanced the monocyte-mediated phagocytosis of CN opsonized with C (CN-absorbed, nonimmune, normal human serum; C-CN). Ingestion of IgG- and C-CN by control and C1q-stimulated monocytes was maximal by 1 h of incubation. The monocyte-mediated enhancement of phagocytosis caused by C1q was paralleled by a proportionate increase in fungicidal activity, an effect which was maximal by 3 h of incubation. Human serum albumin-adherent, control monocytes exhibited only a low level of killing after 3 h of incubation. C1q enhancement was blocked by preincubation of the surfaces with a goat, polyclonal F(ab')2 anti-C1q. This study describes a new cellular function for the cell surface C1q receptor: the enhancement of phagocytosis of a pathogenic organism by monocytes.

C1q acts synergistically with phorbol dibutyrate to activate CR1-mediated phagocytosis by human mononuclear phagocytes.

The adherence of human monocytes and culture-derived macrophages to surfaces coated with complement subcomponent C1q has been previously shown to enhance Fc receptor (FcR)-mediated phagocytosis by these cells. We examined the effects of C1q on C3b/C4b receptor (CR1)-mediated phagocytosis by mononuclear phagocytes. A small percentage of human monocytes cultured in the presence of serum became competent to ingest sheep erythrocytes bearing IgM and C4b (EAC4b). This phagocytic activity was enhanced when these cultured-derived macrophages were adhered to C1q-coated surfaces. However, when cultured in a defined serum-free medium, these cells did not ingest EAC4b, even in the presence of C1q. To investigate this differential responsiveness, we studied the effects of C1q in conjunction with cell-activating agents on CR1 activation. Treatment of serum-free cultured monocytes with phorbol dibutyrate (PDBu), prior to addition of the targets, induced these cells to ingest EAC4b. In addition, when exposed to C1q, both the percentage of these PDBu mononuclear phagocytes ingesting EAC4b and the number of targets ingested increased threefold over the level achieved by macrophages treated with PDBu alone. The chemoattractant N-formyl-methionyl-leucyl-phenylalanine did not activate CR1-mediated phagocytosis and did not substitute for PDBu in causing synergy with C1q. Freshly isolated monocytes adhered to human serum albumin-coated glass slides in the absence or presence of PDBu did not phagocytose EAC4b. Also C1q did not stimulate monocyte CR1-mediated phagocytosis. However, addition of PDBu to cells adherent to the C1q surface triggered phagocytosis of EAC4b. The concentration of PDBu and the time of addition of PDBu relative to addition of the EAC4b targets were found to be important parameters for the achievement of maximal synergy in both the freshly isolated and cultured cell systems. This enhanced phagocytic activity was also seen with cells adhered to the purified collagen-like, pepsin-resistant, fragment of C1q. Since this region was previously shown to interact with C1q surface receptors, it appears that occupancy of this receptor is triggering events contributing to the enhanced cellular function. These experiments suggest that C1q and PDBu promote ingestion via CR1 by different but synergistic mechanisms. These data also demonstrate that the CR1-mediated enhancement of phagocytosis is not specific for FcR-mediated ingestion, but also applies to phagocytosis via CR1.

Inactivation of the small GTP binding protein Rho induces multinucleate cell formation and apoptosis in murine T lymphoma EL4.

The small G-protein Rho regulates the actin microfilament-dependent cytoskeleton. Exoenzyme C3 of Clostridium botulinum ADP-ribosylates Rho at Asn41, a modification that functionally inactivates Rho. Using a Sindbis virus-based transient gene expression system, we studied the role of Rho in murine EL4 T lymphoma cells. We generated a double subgenomic infectious Sindbis virus (dsSIN:C3) recombinant which expressed C3 in >95% of EL4 cells. This intracellular C3 resulted in modification and inactivation of virtually all endogenous Rho. dsSIN:C3 infection led to the formation of multinucleate cells, likely by inhibiting the actin microfilament-dependent step of cytokinesis. Intriguingly, in spite of the inhibition of cytokinesis, karyokinesis continued, with the result that cells containing a nuclear DNA content as high as 16N (eight nuclei) were observed. In addition, dsSIN:C3-mediated inactivation of Rho was a potent activator of apoptosis in EL4 cells. To discern whether the formation of multinucleate cells was responsible for the activation of apoptosis, 5-fluorouracil (5-FUra) was used to induce cell cycle arrest. As expected, EL4 cells treated with 5-FUra were prevented from forming multinucleate cells upon infection with dsSIN:C3. dsSIN:C3 infection, however, still caused marked apoptosis in 5-FUra-treated cells, indicating that this activation of apoptosis was independent of multinucleate cell formation.

Roles of intracellular calcium and NF-kappa B in the Clostridium difficile toxin A-induced up-regulation and secretion of IL-8 from human monocytes.

Clostridium difficile causes an intense inflammatory colitis through the actions of two large exotoxins, toxin A and toxin B. IL-8 is believed to play an important role in the pathophysiology of C. difficile-mediated colitis, although the mechanism whereby the toxins up-regulate the release of IL-8 from target cells is not well understood. In this study, we investigated the mechanisms through which toxin A induces IL-8 secretion in human monocytes. We found that cellular uptake of toxin A is required for the up-regulation of IL-8, an effect that is not duplicated by a recombinant toxin fragment comprising the cell-binding domain alone. Toxin A induced IL-8 expression at the level of gene transcription and this effect occurred through a mechanism requiring intracellular calcium and calmodulin activation. Additionally, the effects of toxin A were inhibited by the protein tyrosine kinase inhibitor genistein, but were unaffected by inhibitors of protein kinase C and phosphatidylinositol-3 kinase. We determined that toxin A activates nuclear translocation of the transcription factors NF-kappa B and AP-1, but not NF-IL-6. NF-kappa B inhibitors blocked the ability of toxin A to induce IL-8 secretion, and supershift analysis indicated that the major isoform of NF-kappa B activated by the toxin is a p50-p65 heterodimer. This study is the first to identify intracellular signaling pathways and transcription factors involved in the C. difficile toxin-mediated up-regulation of IL-8 synthesis and release by target cells. This information should increase our understanding of the pathogenesis of C. difficile colitis and the nature of IL-8 gene regulation as well.

Pre-ligation of CR1 enhances IgG-dependent phagocytosis by cultured human monocytes.

Opsonization of the C3b receptor (CR1) on phagocytic cells with C3b enhances both attachment of targets to the cells and subsequent IgG-dependent ingestion of these targets. To explore mechanisms involved in this increased phagocytosis, we adhered cultured human monocytes to surfaces pre-coated with CR1 ligand or control proteins and quantitated ingestion of sheep E opsonized with IgG alone. Three ligands for CR1 resulted in markedly enhanced phagocytosis of targets when compared individually to a panel of non-ligands, as determined by both the proportion of monocytes ingesting targets (percent phagocytosis) and by the number of targets ingested per 100 monocytes (phagocytic index). The ligands included purified C3b, iC3, and Fab fragments of 1B4, a monoclonal anti-CR1, which resulted in a percent phagocytosis of 56.3 (p less than 0.01), 59.0 (p less than 0.01), and 54.4 (p less than 0.02) and a phagocytic index of 281.2 (p less than 0.01), 281.1 (p less than 0.01), and 247.1 (p less than 0.02), respectively. Control proteins including human serum albumin, hemoglobin, Fab fragments of anti-fibronectin, anti-beta 2 microglobulin, and MOPC 21, and Fc fragments of 1B4 and MOPC 21 produced no significant stimulation of phagocytosis, nor did F(ab')2 fragments of monoclonal anti-CR3, M1/70. CR1-specific augmentation of target ingestion was apparent with monocytes cultured in serum-free medium for 1 to 7 days, but was not seen with freshly elutriated cells. Phagocytosis of unopsonized or IgM-coated targets was minimal. These results suggest that the adherent monocytes are primed by CR1 cross-linking for enhanced FcR-mediated phagocytosis even when the CR1 ligand is not present on the targets. This contrasts with the behavior of CR3, and demonstrated functional divergence between these C3 fragment receptors in the phagocytic process.

Activation of immobilized, biotinylated choleragen AI protein by a 19-kilodalton guanine nucleotide-binding protein.

Cholera toxin catalyzes the ADP-ribosylation that results in activation of the stimulatory guanine nucleotide-binding protein of the adenylyl cyclase system, known as Gs. The toxin also ADP-ribosylates other proteins and simple guanidino compounds and auto-ADP-ribosylates its AI protein (CTA1). All of the ADP-ribosyltransferase activities of CTAI are enhanced by 19-21-kDa guanine nucleotide-binding proteins known as ADP-ribosylation factors, or ARFs. CTAI contains a single cysteine located near the carboxy terminus. CTAI was immobilized through this cysteine by reaction with iodoacetyl-N-biotinyl-hexylenediamine and binding of the resulting biotinylated protein to avidin-agarose. Immobilized CTAI catalyzed the ARF-stimulated ADP-ribosylation of agmatine. The reaction was enhanced by detergents and phospholipid, but the fold stimulation by purified sARF-II from bovine brain was considerably less than that observed with free CTA. ADP-ribosylation of Gsa by immobilized CTAI, which was somewhat enhanced by sARF-II, was much less than predicted on the basis of the NAD:agmatine ADP-ribosyltransferase activity. Immobilized CTAI catalyzed its own auto-ADP-ribosylation as well as the ADP-ribosylation of the immobilized avidin and CTA2, with relatively little stimulation by sARF-II. ADP-ribosylation of CTA2 by free CTAI is minimal. These observations are consistent with the conclusion that the cysteine near the carboxy terminus of the toxin is not critical for ADP-ribosyltransferase activity or for its regulation by sARF-II. Biotinylation and immobilization of the toxin through this cysteine may, however, limit accessibility to Gsa or SARF-II, or perhaps otherwise reduce interaction with these proteins whether as substrates or activator.

GTP but not GDP analogues promote association of ADP-ribosylation factors, 20-kDa protein activators of cholera toxin, with phospholipids and PC-12 cell membranes.

ADP-ribosylation factors (ARFs) are a family of approximately 20-kDa guanine nucleotide-binding proteins initially identified by their ability to enhance cholera toxin ADP-ribosyltransferase activity in the presence of GTP. ARFs have been purified from both membrane and cytosolic fractions. ARF purified from bovine brain cytosol requires phospholipid plus detergent for high affinity guanine nucleotide binding and for optimal enhancement of cholera toxin ADP-ribosyltransferase activity. The phospholipid requirements, combined with a putative role for ARF in vesicular transport, suggested that the soluble protein might interact reversibly with membranes. A polyclonal antibody against purified bovine ARF (sARF II) was used to detect ARF by immunoblot in membrane and soluble fractions from rat pheochromocytoma (PC-12) cell homogenates. ARF was predominantly cytosolic but increased in membranes during incubation of homogenates with nonhydrolyzable GTP analogues guanosine 5'-O-(3-thiotriphosphate), guanylyl-(beta gamma-imido)-diphosphate, and guanylyl-(beta gamma-methylene)-diphosphate, and to a lesser extent, adenosine 5'-O-(3-thiotriphosphate). GTP, GDP, GMP, and ATP were inactive. Cytosolic ARF similarly associated with added phosphatidylserine, phosphatidylinositol, or cardiolipin in GTP gamma S-dependent fashion. ARF binding to phosphatidylserine was reversible and coincident with stimulation of cholera toxin-catalyzed ADP-ribosylation. These observations may reflect a mechanism by which ARF could cycle between soluble and membrane compartments in vivo.

Modulation of FcR function by complement: subcomponent C1q enhances the phagocytosis of IgG-opsonized targets by human monocytes and culture-derived macrophages.

We have investigated the interaction of C1q, a subunit of the first component of complement, with human monocytes and culture-derived macrophages. Adherence of these mononuclear phagocytes to surfaces coated with C1q induced a marked enhancement of the phagocytosis of sheep erythrocytes opsonized with IgG anti-Forssman antibody (EA-IgG). This C1q-mediated enhancement of phagocytosis was dose dependent, and was specifically blocked by pretreatment of the C1q-coated surfaces with F(ab')2 anti-C1q. The augmentation of FcR-mediated phagocytosis by C1q was determined to be a result of the interaction between the C1q and the phagocytic effector cell, and was not due to interaction between the surface-bound C1q and the EA-IgG. Neither resting nor N-formyl-methionyl-leucyl-phenylalanine-stimulated polymorphonuclear leukocytes were induced by C1q to increase FcR-mediated phagocytosis. Experiments conducted with purified fragments of C1q suggest that the C1q phagocytosis enhancement signal resides in the collagen-like tail domain of the molecule. This region is the same portion of the molecule previously shown to interact with the cell surface C1q receptor. Native type I collagen was unable to enhance FcR-mediated phagocytosis by mononuclear phagocytes. It has been demonstrated that C1q can be localized to areas of inflammation, and additionally C1q can be secreted by macrophages in culture. In view of these findings and the results of our present study, we hypothesize that C1q could provide local, direct, and non-opsonic enhancement of phagocytosis by mononuclear phagocytes in areas of infection and inflammation.