Bordetella pertussis filamentous hemagglutinin interacts with a leukocyte signal transduction complex and stimulates bacterial adherence to monocyte CR3 (CD11b/CD18)

Bordetella pertussis, the causative agent of whooping cough, adheres to human monocytes/macrophages by means of a bacterial surface-associated protein, filamentous hemagglutinin (FHA) and the leukocyte integrin, complement receptor 3 (CR3, alpha M beta 2, CD11b/CD18). We show that an FHA Arg-Gly-Asp site induces enhanced B. pertussis binding to monocytes, and that this enhancement is blocked by antibodies directed against CR3. Enhancement requires a monocyte signal transduction complex, composed of leukocyte response integrin (alpha? beta 3) and integrin-associated protein (CD47). This complex is known to upregulate CR3 binding activity. Thus, a bacterial pathogen enhances its own attachment to host cells by coopting a host cell signaling pathway.     

Dissecting human T cell responses against Bordetella species

To identify the minimal structures that may be important for the creation of a synthetic and/or recombinant vaccine against whooping cough, human T cell clones were obtained against Bordetella antigens. Cloned peripheral blood T lymphocytes from an immune donor were grown in IL-2 and tested for proliferation in response to inactivated Bordetella species (B. pertussis, B. parapertussis, and B. bronchiseptica) and mutants deficient for the expression of virulence-associated antigens. All the T cell clones obtained were CD4+8- and recognized specifically the Bordetella antigens when presented by autologous B cells. On the basis of the responsiveness to the whole inactivated bacteria, it was possible to cluster the 12 clones obtained into four groups with the following specificity: (1) filamentous hemagglutinin (FHA); (2) B. pertussis-specific antigens; (3) virulence-associated Bordetella-specific antigens; and (4) nonvirulence-associated Bordetella-specific antigens. Using two new B. pertussis deletion mutants, clone 6 (representative of cluster 1) was found to recognize the COOH terminus of FHA. Furthermore, three out of four clones of cluster 3 were specifically stimulated by the soluble 69-kD protein from the outer membrane of B. pertussis. Surprisingly, none of the twelve clones obtained by stimulation in vitro with whole inactivated bacteria recognized pertussis toxin (PT), which is believed to be the most important protein to be included in an acellular vaccine. However, when a new generation of clones was obtained using soluble PT as the in vitro stimulus, it was observed that 11 clones of this group recognized this antigen. Thus, PT does not seem to be the most representative antigen on the whole inactivated bacteria, although T cell memory against PT exists in a donor who had the disease several years ago.     

Role of adhesin release for mucosal colonization by a bacterial pathogen

Pathogen attachment is a crucial early step in mucosal infections. This step is mediated by important virulence factors called adhesins. To exert these functions, adhesins are typically surface-exposed, although, surprisingly, some are also released into the extracellular milieu, the relevance of which has previously not been studied. To address the role of adhesin release in pathogenesis, we used Bordetella pertussis as a model, since its major adhesin, filamentous hemagglutinin (FHA), partitions between the bacterial surface and the extracellular milieu. FHA release depends on its maturation by the specific B. pertussis protease SphB1. We constructed SphB1-deficient mutants and found that they were strongly affected in their ability to colonize the mouse respiratory tract, although they adhered even better to host cells in vitro than their wild-type parent strain. The defect in colonization could be overcome by prior nasal instillation of purified FHA or by coinfection with FHA-releasing B. pertussis strains, but not with SphB1-producing FHA-deficient strains, ruling out a nonspecific effect of SphB1. These results indicate that the release of FHA is important for colonization, as it may facilitate the dispersal of bacteria from microcolonies and the binding to new sites in the respiratory tract.     

Experimental respiratory infection with Bordetella pertussis in mice: comparison of two methods

The mouse respiratory model is being used increasingly to study the pathogenesis and immunology of Bordetella pertussis infection. Two methods of inoculation, aerosol and intranasal, are routinely used to establish the infection. We compared the two methods of inoculation for reproducibility of infection using quantitative lung cultures and distribution of infection with [35S] methionine labeled bacteria and pulmonary histopathology. Ability to produce a respiratory infection intranasally was related to the inoculum volume; a minimum of 20 microliters was required although considerable variability remained. Lung bacterial counts in identically inoculated mice varied 1,000 fold following intranasal inoculation compared to only 5 fold following aerosol inoculation. Distribution of pulmonary 35S-labeled bacteria varied widely (right lung, 43-84%; left lung 16-57%) following intranasal in comparison to aerosol inoculation (right, 60-68%; left 32-40%). Finally, intranasal inoculation produced a scant, patchy, bronchopneumonia whereas diffuse pathology involving all pulmonary segments was seen following aerosol infection. Due to the superior reproducibility and predictable distribution of infection and pathology, aerosol inoculation is the method of choice for establishing the mouse model of pertussis respiratory infection.     

Pulmonary Alveolar Macrophage DEFENDER AGAINST BACTERIAL INFECTION OF THE LUNG

The rate of ingestion of inhaled bacteria by pulmonary alveolar macrophages is an important determinant of host defense. This parameter was investigated by infecting rats with finely dispersed aerosols bearing Staphylococcus aureus in high concentrations (about 10(s) bacteria/ft(3)/min). These aerosols deposited more than 10(6) bacteria/murine lung. At 0, 2(1/2), and 5 h after infection, bacterial clearance rates were measured in the left lung, and the intracellular or extracellular location of 100 bacteria was determined histologically in the right lung (perfused in situ). The clearance rates at 2(1/2) and 5 h were 44.5% and 76.9%, respectively. The percentages of intracellular bacteria were: 0 h, 54.8%; 2(1/2) h, 87.1%: 5 h, 91.9%. When rats were exposed for 4 h to 2.5 ppm of ozone (O(3)), bacterial clearance did not occur - 15.3%, although 78.7% of the bacteria were intracellular. Clumps of more than 10 bacteria-usually intracellular-were also present. These experiments demonstrate that phagocytic ingestion is an exceedingly rapid process, that in this experimental model the inactivation of inhaled staphylococci results almost entirely from phagocytosis, and that ozone-induced reductions in bacterial clearance are due to severe impairment of intrapulmonary killing mechanisms and minor impairment of bacterial ingestion.     

Receptor analogs and monoclonal antibodies that inhibit adherence of Bordetella pertussis to human ciliated respiratory epithelial cells

The adherence of Bordetella pertussis to human respiratory cilia is critical to the pathogenesis of whooping cough. To explore the development of agents that could interrupt adherence, the structure of the receptor on the ciliary surface was investigated. Using an in vitro adherence assay to human ciliated epithelial cells, galactose, lactose, and complex carbohydrates containing lactose eliminated adherence when preincubated with the bacteria. 10(-2) M galactose eluted adherent bacteria from cilia. B. pertussis and its two purified adhesins bound specifically to natural lactose-containing glycolipids in a TLC assay. mAbs to eukaryotic glycoconjugates with specificity for substituted galactose-glucose moieties blocked adherence when preincubated with ciliated cells. The carbohydrates that serve as receptors for B. pertussis on human cilia are galactose-glucose-containing glycolipids. Receptor analogs and anti-receptor antibodies effectively block adherence of B. pertussis to cilia and thus should be considered candidates for therapeutic intervention against disease.     

IgG subclass responses to Bordetella pertussis filamentous haemagglutinin and pertussis toxin in whooping cough

The IgG subclass response pattern to two major protective antigens of Bordetella pertussis—filamentous haemagglutinin (FHA) and pertussis toxin (PT)—was measured by enzyme-linked immunosorbent assay (ELISA) in paired sera from 46 patients with culture-confirmed whooping cough. Variations in specific subclasses related to antigen and to previous immunization status were noted. The dominant response to both antigens was of the IgG1 subclass and closely followed the total IgG response for both unimmunized children (n = 18) and previously DTP-immunized children and adults (n = 28). Neither IgG2 nor IgG4 to FHA were seen in the unimmunized children but were found in immunized children and adults. An IgG2 response to PT was noted in 70–80% of all patients irrespective of previous immunization status. An observed correlation between IgG2 and neutralizing antibodies to PT was further investigated. It was found that 10 patients without neutralizing antibodies also lacked IgG2 to PT. The study thus revealed differences in IgG subclass responses to two bacterial protein antigens, with an indication that the IgG2 response to PT reflects or correlates to the development of neutralizing antibodies to this toxin. The finding could explain discrepancies between ELISAs and neutralization tests for antibodies to bacterial toxins and have implications for vaccine research.     

Activation of inflammasome signaling mediates pathology of acute P. aeruginosa pneumonia

The respiratory tract is exceptionally well defended against infection from inhaled bacteria, with multiple proinflammatory signaling cascades recruiting phagocytes to clear airway pathogens. However, organisms that efficiently activate damaging innate immune responses, such as those mediated by the inflammasome and caspase-1, may cause pulmonary damage and interfere with bacterial clearance. The extracellular, opportunistic pathogen Pseudomonas aeruginosa expresses not only pathogen-associated molecular patterns that activate NF-κB signaling in epithelial and immune cells, but also flagella that activate the NLRC4 inflammasome. We demonstrate that induction of inflammasome signaling, ascribed primarily to the alveolar macrophage, impaired P. aeruginosa clearance and was associated with increased apoptosis/pyroptosis and mortality in a murine model of acute pneumonia. Strategies that limited inflammasome activation, including infection by fliC mutants, depletion of macrophages, deletion of NLRC4, reduction of IL-1β and IL-18 production, inhibition of caspase-1, and inhibition of downstream signaling in IL-1R– or IL-18R–null mice, all resulted in enhanced bacterial clearance and diminished pathology. These results demonstrate that the inflammasome provides a potential target to limit the pathological consequences of acute P. aeruginosa pulmonary infection.     

Macrophages sense and kill bacteria through carbon monoxide–dependent inflammasome activation

Microbial clearance by eukaryotes relies on complex and coordinated processes that remain poorly understood. The gasotransmitter carbon monoxide (CO) is generated by the stress-responsive enzyme heme oxygenase-1 (HO-1, encoded by Hmox1), which is highly induced in macrophages in response to bacterial infection. HO-1 deficiency results in inadequate pathogen clearance, exaggerated tissue damage, and increased mortality. Here, we determined that macrophage-generated CO promotes ATP production and release by bacteria, which then activates the Nacht, LRR, and PYD domains-containing protein 3 (NALP3) inflammasome, intensifying bacterial killing. Bacterial killing defects in HO-1–deficient murine macrophages were restored by administration of CO. Moreover, increased CO levels enhanced the bacterial clearance capacity of human macrophages and WT murine macrophages. CO-dependent bacterial clearance required the NALP3 inflammasome, as CO did not increase bacterial killing in macrophages isolated from NALP3-deficient or caspase-1–deficient mice. IL-1β cleavage and secretion were impaired in HO-1–deficient macrophages, and CO-dependent processing of IL-1β required the presence of bacteria-derived ATP. We found that bacteria remained viable to generate and release ATP in response to CO. The ATP then bound to macrophage nucleotide P2 receptors, resulting in activation of the NALP3/IL-1β inflammasome to amplify bacterial phagocytosis by macrophages. Taken together, our results indicate that macrophage-derived CO permits efficient and coordinated regulation of the host innate response to invading microbes.     

Increased macrophage infiltration and fractalkine expression in cisplatin-induced acute renal failure in mice

Inflammatory mechanisms contribute to cisplatin-induced acute renal failure (CisARF). Our first aim was to determine renal macrophage infiltration in CisARF. A more than 2-fold increase in CD11b-positive macrophages in the kidney on day 2 preceded the increase in blood urea nitrogen (BUN) and serum creatinine (SCr). Our next aim was to determine the chemoattractant for macrophage infiltration in CisARF. Fractalkine (CX(3)CL1) is expressed on activated endothelial cells and is a potent chemoattractant for macrophages that express its receptor (CX(3)CR1). Immunoblotting showed that whole-kidney CX(3)CL1 expression on days 1, 2, and 3 after cisplatin administration was increased. On immunofluorescence, the intensity of renal endothelial staining of CX(3)CL1 in blood vessels was significantly increased on day 2. Circulating von Willebrand factor (vWF), a measure of systemic endothelial injury, was increased on day 2. Next we determined whether macrophages played an injurious role in CisARF. Macrophages were depleted with injections of liposome-encapsulated clodronate (LEC). LEC resulted in a decrease in renal CD11b-positive macrophages on day 3. However, LEC-treated mice were not protected from CisARF on day 3. To determine the role of CX(3)CR1, both a specific anti-CX(3) CR1 antibody and CX(3) CR1(-/-) mice were used. Administration of the CX(3)CR1 antibody and CX(3) CR1(-/-) mice was not protected against CisARF. In summary, in CisARF, macrophage infiltration in the kidney, CX(3)CL1 expression in whole kidney and blood vessels, and the increase in circulating vWF precede BUN and SCr increase. However, inhibition of macrophage infiltration in the kidney or CX(3)CR1 blockade is not sufficient to prevent CisARF.     

Connective tissue proteins and phagocytic cell function. Laminin enhances complement and Fc-mediated phagocytosis by cultured human macrophages

Brief exposure of culture-derived human macrophages to laminin, a glycoprotein component of all mammalian basement membranes that has a molecular weight of 1,000,000, led to enhancement of subsequent macrophage phagocytosis of EAC4b, EAC3bi, and EAIgG (sheep erythrocytes sensitized with IgG anti-Forssman antibody). This effect on macrophage phagocytosis occurred with both substrate-adherent and fluid phase laminin. Preincubation of macrophages, but not of EAC4b, with laminin led to augmentation of phagocytosis, suggesting that interaction with the phagocytic cell, but not with the opsonized particle, was required for laminin's effect. Laminin-stimulated phagocytosis of EAC4b was blocked entirely by a monoclonal antibody to CR1. Direct comparison of the phagocytic ability of macrophages adherent to laminin- and fibronectin-coated glass slides showed that fibronectin had a somewhat greater enhancing effect on phagocytosis. Nonetheless, the phagocytosis-enhancing effect of laminin was not due to contamination of the purified laminin preparation by fibronectin, since the laminin preparation was free of fibronectin, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay; in addition, laminin-enhanced phagocytosis was decreased in the presence of laminin-specific antibodies. Laminin inhibited macrophage adherence and spreading, but selection of a laminin-binding macrophage subpopulation could not account for the laminin-induced increases in phagocytosis. We hypothesize that interaction with extracellular matrix proteins may represent an important activation stimulus both to the macrophages normally present in the extravascular compartment and to the phagocytic cells that have emigrated from the blood-stream into areas of inflammation.     

Role of complement in mouse macrophage binding of Haemophilus influenzae type b.

Previous in vivo studies demonstrated that clearance of encapsulated Haemophilus influenzae from blood is associated with the deposition of C3 on these bacteria and is independent of the later complement components (C5-C9). Since clearance of encapsulated bacteria is determined by phagocytosis of bacteria by fixed tissue macrophages, we studied the interaction of H. influenzae type b with macrophages in vitro. Organisms bound to macrophages in the presence of nonimmune serum. Binding was not evident in heat-treated serum or in serum from complement depleted animals and was inhibited by F(ab')2 fragments of antibody to C3 and by blockade of the macrophage complement receptor type 3. The majority of organisms bound in the presence of complement alone remained extracellular. Antibody in the form of convalescent serum or an IgG1 monoclonal to type b capsule did not increase the total number of organisms associated with macrophages, but did increase the number of organisms ingested. Furthermore, complement enhanced antibody-mediated ingestion. This in vitro study demonstrates that complement largely mediates binding of H. influenzae to macrophages. This binding may be critical in determining the early clearance of these bacteria from blood and may be an important mechanism of defense in the nonimmune, as well as the immune host.     

Specificity of opsonic antibodies to enhance phagocytosis of Pseudomonas aeruginosa by human alveolar macrophages.

These studies compared the ability of specific secretory IgA (sIgA) and IgG antibodies to promote phagocytosis of viable pseudomonas aeruginosa by human alveolar macrophages. Macrophages were obtained by lung lavage of normal adult smoker and nonsmoker volunteers and were maintained as in vitro cell monolayers. Both immune sIgA and IgG agglutinating antibodies were demonstrated to coat and opsonize viable bacteria, whereas similar nonimmune immunoglobulin preparations did not. When alveolar macrophages were challenged with viable opsonized 14C-labeled Pseudomonas IgG-reacted bacteria were ingested better and killed more readily than sIgA-opsonized organisms. Phagocytic responses were not significantly different between macrophages obtained from smokers and nonsmokers. Although sIgA and IgG antibodies can be found in respiratory secretions and both are undoubtedly important in pulmonary host defense, IgG opsonic antibody was superior in enhancing the uptake of Pseudomonas by in vitro-cultured alveolar macrophages. It may be the more important respiratory antibody for certain bacterial infections.     

Anti-Mac-1 selectively inhibits the mouse and human type three complement receptor

Anti-Mac-1 (M1/70), a rat monoclonal antibody that reacts with mouse and human macrophages, polymorphonuclear leukocytes (PMNL), and natural killer cells, selectively inhibited complement receptor-mediated rosetting by murine macrophages and human PMNL. Preincubation of macrophages with anti-Mac-1 inhibited formation of rosettes with sheep erythrocytes bearing IgM antibody and murine C3 fragments. No inhibition was observed when other monoclonal antibodies that react with macrophages (such as anti-Ly5, anti-H-2, or anti-pan-leukocyte) were tested at 10-fold higher concentrations. Anti-Mac-1 did not affect macrophage Fc receptor-mediated rosetting. Erythrocytes bearing homogeneous human C3 fragments C3b (EC3b) or C3bi (EC3bi) were used to test the specificity of the murine macrophage and human PMNL complement receptor inhibited by anti-Mac-1. In both cases, anti-Mac-1 inhibited CR3-mediated rosetting of EC3bi but not CR1-dependent rosetting of EC3b. The results show that Mac-1 is either identical to CR3 or closely associated with CR3 function. This is one of the first cases in which a monoclonal antibody-defined differentiation antigen has been associated with a specific cell surface function.     

Fas ligand triggers pulmonary silicosis.

We investigated the role of Fas ligand in murine silicosis. Wild-type mice instilled with silica developed severe pulmonary inflammation, with local production of tumor necrosis factor (TNF)-alpha, and interstitial neutrophil and macrophage infiltration in the lungs. Strikingly, Fas ligand-deficient generalized lymphoproliferative disease mutant (gld) mice did not develop silicosis. The gld mice had markedly reduced neutrophil extravasation into bronchoalveolar space, and did not show increased TNF-alpha production, nor pulmonary inflammation. Bone marrow chimeras and local adoptive transfer demonstrated that wild-type, but not Fas ligand-deficient lung macrophages recruit neutrophils and initiate silicosis. Silica induced Fas ligand expression in lung macrophages in vitro and in vivo, and promoted Fas ligand-dependent macrophage apoptosis. Administration of neutralizing anti-Fas ligand antibody in vivo blocked induction of silicosis. Thus, Fas ligand plays a central role in induction of pulmonary silicosis.     

CX3CR1 regulates intestinal macrophage homeostasis, bacterial translocation, and colitogenic Th17 responses in mice

The two most common forms of inflammatory bowel disease (IBD), Crohn's disease and ulcerative colitis, affect approximately 1 million people in the United States. Uncontrolled APC reactivity toward commensal bacteria is implicated in the pathogenesis of the disease. A number of functionally distinct APC populations exist in the mucosal lamina propria (LP) below the intestinal epithelium, but their relative contributions to inflammation remain unclear. Here, we demonstrate in mice important roles for the chemokine receptor CX3CR1 in maintaining LP macrophage populations, preventing translocation of commensal bacteria to mesenteric lymph nodes (mLNs), and limiting colitogenic Th17 responses. CX3CR1 was found to be expressed in resident LP macrophages (defined as CD11b(+)F4/80(+)) but not DCs (defined as CD11c(+)CD103(+)). LP macrophage frequency and number were decreased in two strains of CX3CR1-knockout mice and in mice deficient in the CX3CR1 ligand CX3CL1. All these knockout strains displayed markedly increased translocation of commensal bacteria to mLNs. Additionally, the severity of DSS-induced colitis was dramatically enhanced in the knockout mice as compared with controls. Disease severity could be limited by either administration of neutralizing IL-17A antibodies or transfer of CX3CR1-sufficient macrophages. Our data thus suggest key roles for the CX3CR1/CX3CL1 axis in the intestinal mucosa; further clarification of CX3CR1 function will likely direct efforts toward therapeutic intervention for mucosal inflammatory disorders such as IBD.     

微小RNA-191抑制巨噬细胞EGR1介导小鼠巨噬细胞对脓毒症的影响

目的探讨微小RNA-191(miR-191)抑制巨噬细胞早期生长反应蛋白1(EGR1)介导小鼠巨噬细胞对脓毒症的影响。方法无特定病原级雌性BALB/c小鼠,构建脓毒症模型后分为对照组(n=10)和miR-191组(n=10)。对照组经腹腔注射10 mg/kg的0.9%氯化钠溶液,miR-191组经腹腔注射10 mg/kg的miR-191质粒。观察48 h记录小鼠生存情况,采用腹腔灌洗法收集腹腔灌洗液检测细菌负荷,苏木精伊红染色法观察肺脏组织变化。培养腹腔巨噬细胞并进行体外实验,分为A组和B组,A组加入miR-191质粒,B组加入等体积0.9%氯化钠溶液。检测A组和B组EGR1含量和细菌数目。结果miR-191组小鼠生存率高于对照组,腹腔和血液细菌负荷数小于对照组,差异有统计学意义(P<0.05)。对照组小鼠肺泡结构紊乱,肺泡间隔明显增宽,肺间质有明显充血水肿,部分肺不张,有大量中性粒细胞浸润;miR-191组小鼠肺泡结构基本完整,肺泡间隔增宽,肺间质充血水肿明显改善,中性粒细胞浸润明显减少。体外实验结果显示,A组小鼠巨噬细胞EGR1水平低于B组,细胞内细菌存活数低于B组,差异有统计学意义(P<0.05)。结论miR-191通过抑制EGR1表达,有效降低脓毒症小鼠氧化应激反应,减轻肺组织病理损伤,提高细菌清楚能力,这可为临床治疗脓毒症提供参考。     

Sero epidemiology of Bordetella pertussis immune responses in a healthy population in northern Greece

A seroepidemiological study was conducted on a representative sample of the northern Greek population (healthy individuals, age range=1 day to 80 years) to assess the prevalence of antibodies to pertussis toxin (PT) and filamentous hemagglutinin (FHA). Antibody concentrations were significantly elevated with age (analysis of variance (ANOVA), P<0.001). In addition, a significant increase in antibody levels was detected in subjects >50 years old compared to children aged 5-10 years (post-hoc Scheffe analysis, P=0.007). These data suggest that pertussis occurs frequently in Greek adults, and that sometimes a fifth booster vaccine dose is not given after the second year of life. Routine revaccination with the acellular vaccine for children >4 years of age, adolescents, and adults should be considered in order to ensure effective protection of the whole population.     

Bleomycin-induced pulmonary fibrosis in hamsters. An alveolar macrophage product increases fibroblast prostaglandin E2 and cyclic adenosine monophosphate and suppresses fibroblast proliferation and collagen production

Bleomycin-induced pulmonary fibrosis in hamsters is associated with collagen accumulation that results from increased lung collagen synthesis rates. However, 1-2 wk after intratracheal instillation of bleomycin, lung collagen synthesis rates decline toward control values. To evaluate the potential role of the bronchoalveolar macrophage in regulating lung collagen production, we studied the effects of macrophages from normal and bleomycin-treated hamsters upon fibroblasts in vitro. We observed: (a) Medium from macrophage cultures decreased fibroblast [3H]thymidine incorporation and nondialyzable [3H]hydroxyproline production in a dose-dependent manner. Fibroblast cell counts were decreased in exposed cultures, and fibroblast viability was unchanged. Procollagen prolyl hydroxylation and prolyl-transfer RNA-specific activity were not altered by macrophage medium; this indicates that [3H]hydroxyproline reflects collagen production rate under the experimental conditions. (b) The suppressive effect of macrophage medium was selective for collagen since collagen production decreased more than noncollagen protein production. (c) Medium from bleomycin-treated hamster macrophages suppressed fibroblast proliferation and collagen production to a greater degree than medium from normal hamster macrophages. (d) Fibroblast suppression by macrophage medium was associated with increased fibroblast endogenous prostaglandin E2 production and intracellular cyclic AMP (cAMP). (e) Incubation of fibroblasts with indomethacin before exposure completely inhibited prostaglandin E2 production and increases in cAMP, and eliminated suppression of fibroblast proliferation and collagen production. The macrophage-derived suppressive factor has an apparent molecular weight of 20,000-30,000 and is heat stable. It does not appear to be species restricted since both hamster and human lung fibroblasts are similarly suppressed. It is at least in part preformed in macrophages obtained by lavage, but its production can also be stimulated in vitro. We concluded that alveolar macrophages release a product that stimulates endogenous fibroblast prostaglandin E2 production and cAMP formation with resultant suppression of fibroblast proliferation and collagen production. Enhanced release of suppressive factor by macrophages during a time when lung collagen production is declining in bleomycin-induced pulmonary fibrosis suggests that macrophages may limit collagen accumulation in pulmonary fibrosis.     

The role of complement receptors in tumor cell destruction.

Murine peritoneal macrophages exuding early after stimulation with the activators of the alternative complement pathway are able to destroy some of the tumor cells. This destruction is inhibited by anti-Mac-1 and anti-C3. The tumor cell killing needs longer incubation time (> 15 hr), and anti-Mac-1 affects the reaction even if it were put into the reaction mixture at later time of incubation. The results suggest that complement produced by macrophage is deposited onto target cells, and the complement binding targets interact with macrophages through the complement receptor type 3 (CR3), which leads to the cell destruction. It is known that fully activated macrophages kill the tumor cells with the aid of antibody (ADCC) or lectin, but we show here another route of tumor cell destruction, that is, some sort of incompletely activated macrophages can kill some type of tumor cells in cooperation with endogenous complement and complement receptors (CR3) without participation of antibody.