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Macrophages are mononuclear phagocytes that are widely distributed throughout the body. These cells can contribute to development and homeostasis and participate in innate and adaptive immune responses. The physiology of macrophages can vary tremendously depending on the environment in which they reside and the local stimuli to which they are exposed. Macrophages are prodigious secretory cells, and in that role can promote and regulate immune responses and contribute to autoimmune pathologies. Macrophages are highly phagocytic, and in this capacity have long been considered to be essential immune effector cells. The important roles of macrophages in maintaining homeostasis and in contributing to tissue remodeling and wound healing is sometimes overlooked because of their vital role in host defense., (Copyright 2008 by John Wiley & Sons, Inc.)

Our understanding of cell mediated immunity (CMI) has revealed the importance of activated macrophages as key immune effector cells. Over the past decade, we have come to realize that macrophages exhibit remarkable plasticity, and different populations of macrophages with distinct physiologies can develop in response to different stimuli. In fact, it is likely that the number of different macrophage populations that can arise may be as diverse as the activating stimuli that induce them. Some of these stimuli can instruct macrophages to kill microbes (classical activation), lay down extracellular matrix components to promote wound healing (alternative activation), or secrete anti-inflammatory cytokines to terminate inflammation (regulatory macrophages). New ways to biochemically identify these cells have led to a better understanding of the heterogeneity of activated macrophages. As our understanding of the various macrophage populations increases, so does the potential for therapeutic intervention based on targeting specific populations of activated macrophages., (Copyright 2008 by John Wiley & Sons, Inc.)

Macrophage CSF (M-CSF) regulates monocyte differentiation, activation, and foam cell formation. We have observed that it is elevated in human pulmonary alveolar proteinosis (PAP) and in the GM-CSF knockout mouse, a murine model for PAP. A potential regulator of M-CSF, peroxisome proliferator-activated receptor-gamma (PPARgamma), is severely deficient in both human PAP and the GM-CSF knockout mouse. To investigate the role of PPARgamma in alveolar macrophage homeostasis, we generated myeloid-specific PPARgamma knockout mice using the Lys-Cre method to knock out the floxed PPARgamma gene. Similar to the GM-CSF-deficient mouse, absence of alveolar macrophage PPARgamma resulted in development of lung pathology resembling PAP in 16-wk-old mice, along with excess M-CSF gene expression and secretion. In ex vivo wild-type alveolar macrophages, we observed that M-CSF itself is capable of inducing foam cell formation similar to that seen in PAP. Overexpression of PPARgamma prevented LPS-stimulated M-CSF production in RAW 264.7 cells, an effect that was abrogated by a specific PPARgamma antagonist, GW9662. Use of proteasome inhibitor, MG-132 or a PPARgamma agonist, pioglitazone, prevented LPS-mediated M-CSF induction. Using chromatin immunoprecipitation, we found that PPARgamma is capable of regulating M-CSF through transrepression of NF-kappaB binding at the promoter. Gel-shift assay experiments confirmed that pioglitazone is capable of blocking NF-kappaB binding. Taken together, these data suggest that M-CSF is an important mediator of alveolar macrophage homeostasis, and that transcriptional control of M-CSF production is regulated by NF-kappaB and PPARgamma.

In endothelial cells, NF-kappaB is an important intracellular signaling molecule by which changes in wall shear stress are transduced into the nucleus to initiate downstream endothelial nitric oxide synthase (NOS3) gene expression. We investigated whether NF-kappa light-chain gene enhancer in B cells 1 (NFKB1) promoter polymorphism ((-94)NFKB1 I/D, where I is the insertion allele and D is the deletion allele) was associated with 1) NOS3 gene expression in endothelial cells under physiological levels of unidirectional laminar shear stress (LSS) and 2) endothelial function in prehypertensive and stage I hypertensive individuals before and after a 6-mo supervised endurance exercise intervention. Competitive EMSAs revealed that proteins present in the nuclei of endothelial cells preferentially bound to the I allele NFKB1 promoter compared with the D allele. Reporter gene assays showed that the I allele promoter had significantly higher activity than the D allele. In agreement with these observations, homozygous II genotype cells had higher p50 expression levels than homozygous DD genotype cells. Cells with the homozygous II genotype showed a greater increase in NOS3 protein expression than did homozygous DD genotype cells under LSS. Functional experiments on volunteers confirmed higher baseline reactive hyperemic forearm blood flow, and, furthermore, the subgroup analysis revealed that DD homozygotes were significantly less prevalent in the exercise responder group compared with II and ID genotypes. We conclude that the (-94)NFKB1 I/D promoter variation contributes to the modulation of vascular function and adaptability to exercise-induced flow shear stress, most likely due to differences in NFKB1 gene transactivity.

The term "monocyte" implies a single, homogenous population of cells with uniform physiology. Recent evidence from a number of laboratories indicates that it is likely that blood monocytes may consist of several subpopulations of cells, which differ in size, nuclear morphology, granularity, and functionality. The aim of this review is to give a summary of the new findings in the emerging field of monocyte heterogeneity. We provide a short description of the differentiation patterns of blood monocyte subpopulations, with an emphasis on how these subpopulations can be influenced by infection. We provide a comparison among the main monocyte subpopulations in humans, mice, and rats and illustrate some of the common features of these cells and some of the important interspecies distinctions. We will also discuss the bone marrow precursors of these cells and the differentiation patterns of these subsets in different tissues in response to infection. Most of the data about monocyte trafficking during infection are necessarily derived from murine models, and comparisons between mouse and man must be made with caution. However, these models may provide interesting springboards to permit us to speculate about the topic of monocyte heterogeneity in humans.

Leishmanization is the inoculation of live Leishmania into the host to vaccinate against subsequent infections. This approach has been largely discontinued due to safety concerns. We have previously shown that combining CD40 ligand (CD40L) with Leishmania antigen preferentially induces a type 1 immune response and provides some protection to vaccinated mice (G. Chen, P. A. Darrah, and D. M. Mosser, Infect. Immun. 69:3255-3263, 2001). In the present study, we developed transgenic L. major organisms which express and secrete the extracellular portion of CD40L (L. major CD40LE). We hypothesized that these organisms would be less virulent but more immunogenic than wild-type organisms and therefore be more effective at leishmanization. Transgenic parasites expressing CD40L mRNA and protein were developed. BALB/c mice infected with these parasites developed significantly smaller lesions containing fewer parasites than animals infected with wild-type organisms. Infection of resistant C57BL/6 mice with low doses of transgenic parasites induced a significant amount of protection against subsequent high-dose infection with wild-type organisms. These results demonstrate that transgenic organisms expressing CD40L are less virulent than wild-type organisms while retaining full immunogenicity.

Transgenic Leishmania parasites that encode the murine chemokine monocyte chemoattractant protein 1 (MCP-1) were generated. These parasites transcribed MCP-1 mRNA and secreted MCP-1 protein. Infection of BALB/c, C57BL/6, or MCP-1 knockout (KO) mice with these parasites resulted in minimal lesion development with fewer parasites in the infected foot, lymph node, and spleen compared to wild-type-infected mice. In contrast, transgenic parasites caused substantial lesions with relatively high numbers of parasites in CC chemokine receptor 2 (CCR2) KO mice, indicating that the parasites are viable and healthy and that the lack of lesion development is CCR2 dependent. Prior infection of mice with transgenic parasites offered no protection to subsequent wild-type L. major challenge, suggesting that the transgenic parasites are controlled by an early innate immune response. Consistent with innate immunity, flow cytometry of cells from the ears of mice infected with transgenic parasites revealed an increase in the number of CCR2-positive macrophages by day 7 postinfection. The enumeration of transgenic parasites in ear lesions demonstrated a significant reduction in parasite numbers, which coincided with the increased CCR2-positive macrophage migration. CCR2-positive macrophages isolated from ears of mice infected with transgenic parasites contained virtually no parasites. In vitro studies revealed that optimal parasite killing required the recruitment of CCR2-positive macrophages, followed by stimulation with a combination of both MCP-1 and gamma interferon (IFN-gamma). This work suggests that the parasite-derived MCP-1 can recruit a restrictive population of CCR2-positive macrophages into lesions that can be optimally stimulated by MCP-1 and IFN-gamma to efficiently kill Leishmania parasites.

IL-10 is a critical cytokine in determining host susceptibility to Leishmania spp. We previously demonstrated that macrophage-derived IL-10 could contribute to disease exacerbation, but the mechanisms whereby Leishmania infections led to IL-10 induction were not fully understood. In this study, we demonstrated that infection of macrophages with Leishmania amazonensis amastigotes led to the activation of the MAPK, ERK1/2. This activation was required, but not sufficient for IL-10 induction. In addition to ERK activation, an inflammatory stimulus, such as low m.w. hyaluronic acid from the extracellular matrix, must also be present. The combination of these two signals resulted in the superinduction of IL-10. We also demonstrated that IgG on the surface of Leishmania amastigotes was required to achieve maximal IL-10 production from infected macrophages. Surface IgG engages macrophage FcgammaR to induce ERK activation. Macrophages lacking FcgammaR, or macrophages treated with an inhibitor of spleen tyrosine kinase, the tyrosine kinase that signals via FcgammaR, failed to activate ERK and consequently failed to produce IL-10 following infection with Leishmania amastigotes. We confirmed that ERK1/2 activation led to the phosphorylation of histone H3 at the IL-10 promoter, and this phosphorylation allowed for the binding of the transcription factor, Sp1, to the IL-10 promoter. Finally, the administration of U0126, an inhibitor of ERK activation, to infected mice resulted in decreased lesion progression with reduced numbers of parasites in them. Thus, our findings reveal an important role of MAPK, ERK signaling in the pathogenesis of Leishmania infection.

We generated three populations of macrophages (Mphi) in vitro and characterized each. Classically activated Mphi (Ca-Mphi) were primed with IFN-gamma and stimulated with LPS. Type II-activated Mphi (Mphi-II) were similarly primed but stimulated with LPS plus immune complexes. Alternatively activated Mphi (AA-Mphi) were primed overnight with IL-4. Here, we present a side-by-side comparison of the three cell types. We focus primarily on differences between Mphi-II and AA-Mphi, as both have been classified as M2 Mphi, distinct from Ca-Mphi. We show that Mphi-II more closely resemble Ca-Mphi than they are to AA-Mphi. Mphi-II and Ca-Mphi, but not AA-Mphi, produce high levels of NO and have low arginase activity. AA-Mphi express FIZZ1, whereas neither Mphi-II nor Ca-Mphi do. Mphi-II and Ca-Mphi express relatively high levels of CD86, whereas AA-Mphi are virtually devoid of this costimulatory molecule. Ca-Mphi and Mphi-II are efficient APC, whereas AA-Mphi fail to stimulate efficient T cell proliferation. The differences between Ca-Mphi and Mphi-II are more subtle. Ca-Mphi produce IL-12 and give rise to Th1 cells, whereas Mphi-II produce high levels of IL-10 and thus, give rise to Th2 cells secreting IL-4 and IL-10. Mphi-II express two markers that may be used to identify them in tissue. These are sphingosine kinase-1 and LIGHT (TNF superfamily 14). Thus, Ca-Mphi, Mphi-II, and AA-Mphi represent three populations of cells with different biological functions.

NF-kappaB/Rel is a family of transcription factors whose activation has long been linked to the production of inflammatory cytokines. Here, we studied NF-kappaB signaling in the regulation of the anti-inflammatory cytokine, interleukin-10 (IL-10). We identified a role for a single NF-kappaB family member, NF-kappaB1 (p50), in promoting the transcription of IL-10. The NF-kappaB ciselement on IL-10 proximal promoter was located to -55/-46, where p50 can homodimerize and form a complex with the transcriptional co-activator CREB-binding protein to activate transcription. The other Rel family members appear to play a negligible role in IL-10 transcription. Mice lacking p50 were more susceptible to lethal endotoxemia, and macrophages taken from p50-/- mice exhibit skewed cytokine responses to lipopolysaccharide, characterized by decreased IL-10 and increased tumor necrosis factor and IL-12. Taken together, our studies demonstrate that NF-kappaB1 (p50) homodimers can be transcriptional activators of IL-10. The reciprocal regulation of pro- and anti-inflammatory cytokine production by NF-kappaB1 (p50) may provide potential new ways to manipulate the innate immune response.

To gain insight into the molecular mechanism(s) whereby macrophages produce large amounts of IL-10, we analyzed IL-10 gene expression and temporally correlated it with modifications to chromatin associated with the IL-10 promoter. In resting cells, which make essentially no cytokines, the IL-10 promoter is associated with histones containing little or no detectable modifications. Macrophages stimulated in the presence of immune complexes begin to produce high levels of IL-10 pre-mRNA transcripts within minutes of stimulation. Coincident with this transcription was a rapid and dynamic phosphorylation of histone H3 at specific sites in the IL-10 promoter. Histone phosphorylation was closely followed by the binding of transcription factors to the IL-10 promoter. Blocking the activation of ERK prevented histone phosphorylation and transcription factor binding to the IL-10 promoter. In contrast to histone phosphorylation, the peak of histone acetylation at this promoter did not occur until after transcription had peaked. Inhibition of histone deactylase did not alter IL-10 gene expression, suggesting that phosphorylation but not acetylation was the proximal event responsible for IL-10 transcription. Our findings reveal a rapid and well-orchestrated series of events in which ERK activation causes a rapid and transient phosphorylation of histone H3 at specific regions of the IL-10 promoter, resulting in a transient exposure of the IL-10 promoter to the transcription factors that bind there. This exposure is essential for the efficient induction of IL-10 gene expression in macrophages. To our knowledge, this represents a unique way in which the expression of a cytokine gene is regulated in macrophages.

We have previously demonstrated that macrophages stimulated in the presence of immune complexes produce high levels of IL-10. We now examine the mechanism of IL-10 superinduction. We report that the enhanced production of IL-10 correlates with a rapid and enhanced activation of two MAPKs, ERK and p38. The inhibition of either ERK or p38 prevented IL-10 induction, indicating that both MAPKs were required for IL-10 synthesis. By chromatin immunoprecipitation assay, we demonstrate that activation of ERK leads to the phosphorylation of serine 10 on histone H3 at the il-10 gene, making the promoter more accessible to transcription factors generated in response to p38 activation. Inhibition of ERK activation prevented histone modifications, and decreased the binding of Sp1 and STAT3 to the IL-10 promoter. We conclude that the activation of ERK following FcgammaR ligation leads to a remodeling of the chromatin at the il-10 locus, making it more accessible to transcription factors. The rapid and transient regulation of transcription factor accessibility to the IL-10 promoter by MAPK activation represents a novel way that the production of this cytokine is regulated.

Background: The Leishmania promastigote-macrophage interaction occurs through the association of multiple receptors on the biological membrane surfaces. The success of the parasite infection is dramatically dependent on this early interaction in the vertebrate host, which permits or not the development of the disease. In this study we propose a novel methodology using flow cytometry to study this interaction, and compare it with a previously described "in vitro" binding assay., Methods: To study parasite-macrophage interaction, peritoneal macrophages were obtained from 4 dogs and adjusted to 3 x 10(6) cells/mL. Leishmania (Leishmania) chagasi parasites (stationary-phase) were adjusted to 5 x 10(7) cells/mL. The interaction between CFSE-stained Leishmania chagasi and canine peritoneal macrophages was performed in polypropylene tubes to avoid macrophage adhesion. We carried out assays in the presence or absence of normal serum or in the presence of a final concentration of 5% of C5 deficient (serum from AKR/J mice) mouse serum. Then, the number of infected macrophages was counted in an optical microscope, as well as by flow citometry. Macrophages obtained were stained with anti-CR3 (CD11b/CD18) antibodies and analyzed by flow citometry., Results: Our results have shown that the interaction between Leishmania and macrophages can be measured by flow cytometry using the fluorescent dye CFSE to identify the Leishmania, and measuring simultaneously the expression of an important integrin involved in this interaction: the CD11b/CD18 (CR3 or Mac-1) beta2 integrin., Conclusion: Flow cytometry offers rapid, reliable and sensitive measurements of single cell interactions with Leishmania in unstained or phenotypically defined cell populations following staining with one or more fluorochromes.

We examined the role of immunoglobulin (Ig)G antibodies in mediating host defense to the intracellular parasite, Leishmania. We show that IgG not only fails to provide protection against this intracellular pathogen, but it actually contributes to disease progression. The J(H) strain of BALB/c mice, which lack IgG because they have a targeted deletion in the Ig heavy chain (J) locus, were more resistant to infection with Leishmania major than were normal BALB/c mice. However, the passive administration of anti-Leishmania IgG caused J(H) mice to develop large lesions containing high numbers of parasites. Antibody administration correlated with an increase in interleukin (IL) 10 production in lesions, and blocking the murine IL-10 receptor prevented antibody-mediated disease exacerbation. In human patients with active visceral leishmaniasis, high IgG levels are predictive of disease. Patients with ongoing disease had high IgG antibody titers and no delayed-type hypersensitivity (DTH) responses to Leishmania antigens. This pattern was reversed upon disease resolution after treatment, resulting in a decrease in total IgG, which was accompanied by a progressive increase in DTH responsiveness. We conclude that IgG can cause a novel form of immune enhancement due to its ability to induce IL-10 production from macrophages.

We examined innate immune responses to the intracellular bacterium Rhodococcus equi and show that infection of macrophages with intact bacteria induced the rapid translocation of NF-kappa B and the production of a variety of proinflammatory mediators, including TNF, IL-12, and NO. Macrophages from mice deficient in MyD88 failed to translocate NF-kappa B and produced virtually no cytokines in response to R. equi infection, implicating a TLR pathway. TLR4 was not involved in this response, because C3H/HeJ macrophages were fully capable of responding to R. equi infection, and because RAW-264 cells transfected with a dominant negative form of TLR4 responded normally to infection by R. equi. A central role for TLR2 was identified. A TLR2 reporter cell was activated by R. equi, and RAW-264 cells transfected with a dominant negative TLR2 exhibited markedly reduced cytokine responses to R. equi. Moreover, macrophages from TLR2(-/-) mice exhibited diminished cytokine responses to R. equi. The role of the surface-localized R. equi lipoprotein VapA (virulence-associated protein A), in TLR2 activation was examined. Purified rVapA activated a TLR2-specific reporter cell, and it induced the maturation of dendritic cells and the production of cytokines from macrophages. Importantly, TLR2(-/-)-deficient but not TLR4(-/-)-deficient mice were found to be compromised in their ability to clear a challenge with virulent R. equi. We conclude that the efficient activation of innate immunity by R. equi may account for the relative lack of virulence of this organism in immunocompetent adults.

We have previously reported that the ligation of FcgammaRs on activated macrophages affected their production of cytokines and their ability to influence T cell activation. Dendritic cells (DC) are important APCs that also express FcgammaR. In the present work, we sought to determine whether DC responded to immune complexes in a manner similar to macrophages. We confirmed that activated murine DC produced IL-12, and, as a result, induced naive T cells to produce primarily IFN-gamma upon stimulation. However, DC activated in the presence of immune complexes shut off their production of IL-12p70 and induced a Th2-like cytokine response. Thus, DC respond to immune complexes by altering their cytokine production, which, in turn, influences T cell responses. A DC transfer experiment was performed to determine the extent that APC exposure to immune complexes could influence adaptive immune responses. Vaccination of mice with Ag, along with DC that were activated in the presence of immune complexes, resulted in higher levels of Ag-specific IgG1 Ab, relative to mice that were vaccinated with activated DC and Ag alone. The mechanism by which DC altered their cytokine production in response to immune complexes was different from macrophages. Macrophages down-regulated the transcription of both the p40 and p35 subunits of IL-12, whereas DC decreased only p35 expression. We conclude that APCs expressing FcgammaR on their surface can respond to immune complexes by shutting off IL-12 biosynthesis, to prevent the Th1-type T cell biasing that normally accompanies innate immune activation.

Activated macrophages were used as antigen presenting cells (APCs) to determine the extent to which these APCs could influence an adaptive immune response. We show that activated macrophages induced a strong polarized Th1-like T cell response that was predominated by IFN-gamma. However, when antigen was targeted to Fcgamma receptors on these macrophages, their phenotype changed, and they now induced a T cell response that was predominated by IL-4. The initial biasing by activated macrophages toward a Th1-like response was a result of activation of the innate immune response, as macrophages from MyD88(-/-) mice failed to produce Th1-inducing cytokines. The reversal of the Th1 biasing was a result of FcgammaR ligation, as macrophages lacking the FcR common gamma chain failed to reverse this biasing. To show that this biasing could occur in vivo, mice were injected with activated macrophages or activated macrophages whose FcgammaR had been ligated with an irrelevant immune complex. Mice injected with FcgammaR-ligated macrophages made more antibody than those receiving conventionally activated macrophages, and the antibody was predominantly of the IgG1 isotype. These studies demonstrate that FcgammaR ligation on activated macrophages can change the phenotype of these APCs to cells that preferentially drive a Th2-like response. We have termed these cells type 2 activated macrophages.

Activated macrophages are a critical component of our antimicrobial armamentarium. Unfortunately, the lipid mediators and free radicals that these cells produce are not only toxic to potential pathogens, but also to the host. Thus the modulation of these activities can mitigate an overzealous immune response and thereby prevent host cell injury. Two families of receptor tyrosine kinases (RTK) in macrophages, the RON/STK and the Tyro3 families of protein kinases, will be examined in this review with an emphasis on their roles in modulating the effector functions of activated macrophages. Both families of receptors are capable of down-regulating the inflammatory response of macrophages to lipopolysaccharide, and both families of RTK's are structurally related. An analysis of the intracellular domains of RON/STK and Tyro3 reveal a common multi-substrate binding site, which can recruit common signaling molecules such as growth factor receptor bound 2 (Grb2) and phosphatidylinositol 3-kinase (PI3-K). The observations relating to a modulation of macrophage effector mechanisms by these receptors open unexplored avenues for the development of pharmacological immunomodulators with the potential to exploit elements of this common pathway.

An immune response can deviate toward either a Th1- or Th2-like response. In this work we examine the contribution that activated macrophages and IgG Abs make toward this deviation. The use of activated macrophages as APCs resulted in a strong polarized T cell response that was predominated by IFN-gamma. However, when Ag was targeted to FcgammaRs on these macrophages, the T cell response was reversed and biased toward a Th2-like response. This Th2-like phenotype was stable and was retained when the T cells were subsequently restimulated under nonbiasing conditions. The T cell biasing and its reversal via FcgammaR was also observed in vivo. Mice vaccinated with IgG-opsonized OVA made high levels of IgG Ab of the IgG1 isotype. These studies demonstrate that the ligation of FcgammaR on activated macrophages can reverse the Th1 biasing that occurs as a result of innate immune responses to microbial products.

Macrophages respond to bacterial products by releasing a large array of inflammatory mediators. We demonstrate that, in the presence of IgG immune complexes, macrophages produce high levels of IL-10 and virtually no IL-12, when they are exposed to bacterial products. The production of IL-10 by these cells can dampen innate inflammatory responses to microbial products, such as LPS. This alteration in macrophage cytokine production can also influence an adaptive immune response, preferentially inducing Th2-type immunity. Thus, immune complexes change the physiology of activated macrophages, converting them to anti-inflammatory cells that induce Th2-like immune responses. We have termed these cells type II activated macrophages.

The genetic background of mice infected with Leishmania major determines the response to infection, resulting in a resistant or susceptible phenotype. Susceptible mice develop a T-helper type 2 (Th2)-type immune response following infection distinguished by the development of interleukin (IL)-4 secreting T cells in the lymph node and spleen. In SJL mice, which normally heal L. major lesions, subtoxic doses of mercury induce an autoimmune syndrome characterized by an expansion of Th2 cells. In this study, we examined the effect of mercury administration on the outcome of L. major infection in SJL mice. We show that subtoxic doses of mercuric chloride (HgCl2) exacerbate disease outcome in SJL mice resulting in increased footpad swelling and increased parasite burdens. Furthermore, the effects of HgCl2 treatment on resistance to L. major are time-dependent. The nonhealing phenotype was observed only if mice had been treated with HgCl2 prior to L. major infection for at least 1 week, a timepoint at which mice treated with HgCl2 alone had increased splenocyte IL-4 production. HgCl2 treatment also increased production of serum immunoglobulin (Ig)E and IgG1, two IL-4 dependent isotypes. These results show that HgCl2 treatment enhances the susceptibility to L. major in SJL mice, consistent with the induction of host Th2 parameters. These findings have implications for the role of mercury contamination in areas of endemic leishmaniasis.

Our laboratory has previously demonstrated that the ligation of phagocytic receptors on macrophages can influence cytokine production. In this study, we examine the cytokine responses to multiple inflammatory stimuli following FcgammaR ligation. Macrophages were stimulated in vitro with LPS, lipoteichoic acid, CD40 ligand, or low molecular mass hyaluronic acid. All of these stimuli were proinflammatory in character, inducing the production of high levels of IL-12, but only modest amounts of IL-10. The coligation of FcgammaR along with these stimuli resulted in an anti-inflammatory profile, abrogating IL-12 production and inducing high levels of IL-10. The modulation of these two cytokines occurred by two independent mechanisms. Whereas the abrogation of IL-12 biosynthesis was a property shared by several macrophage receptors, the induction of IL-10 was specific to the FcgammaR. The biological relevance of these observations was examined in murine models of endotoxemia, in which FcgammaR ligation induced the rapid production of IL-10 and prevented IL-12 synthesis. Mice could be passively immunized with Abs to LPS to reverse inflammatory cytokine production, and the transfer of macrophages whose FcgammaR had been ligated could rescue mice from lethal endotoxemia. Thus, the ligation of the macrophage FcgammaR can be exploited to prevent inappropriate inflammatory cytokine responses.

CD40 ligand (CD40L) is a potent inducer of interleukin-12 (IL-12) production from macrophages and dendritic cells. We show that combining CD40L with antigen derived from Leishmania is an effective way to preferentially induce type 1 immune responses to the antigen and to vaccinate mice against subsequent challenge with virulent organisms. Mice vaccinated in this way had smaller lesions, with more than 1,000-fold fewer parasites within them. To improve the efficiency of CD40L-induced immunopotentiation, we attempted to specifically direct CD40L to macrophages. We developed transfected cells expressing CD40L and a single Leishmania antigen, gp63. These cells bound efficiently to macrophages and induced robust IL-12 production. Vaccination with these cotransfected cells provided a significant degree of protection against challenge with virulent organisms. CD40L was also adsorbed to the surface of virulent Leishmania. These organisms induced only modest lesions in genetically susceptible mice, and the lesions had an average of 10(5)-fold fewer organisms within them relative to control mice. These studies suggest that CD40L could be exploited to improve vaccines against intracellular pathogens, especially those organisms that reside within cells expressing CD40 on their surface.

Ligating Fc gamma R on macrophages results in suppression of IL-12 production. We show that Fc gamma R ligation selectively down-regulates IL-12 p40 and p35 gene expression at the level of transcription. The region responsive to this inhibition maps to the Ets site of the p40 promoter. PU.1, IFN consensus sequence binding protein, and c-REL: form a complex on this element upon macrophage activation. Receptor ligation abolishes the binding of this PU.1-containing activation complex, and abrogates p40 transcription. A dominant-negative construct of PU.1 diminishes IL-12 p40 promoter activity and endogenous IL-12 p40 protein secretion. Thus, the specificity of IL-12 down-regulation following receptor ligation lies in the inhibition of binding of a PU.1-containing complex to the Ets site of the IL-12 promoter. These findings provide evidence demonstrating for the first time the importance of PU.1 in the transcriptional regulation of IL-12 gene expression.

The macrophage receptors for the Fc portion of immunoglobulin G (FcgammaR) have long been known to mediate a variety of effector functions that are vital to the adaptive immune response. Recent studies, however, have begun to stress potential regulatory roles that these receptors can play in modulating immune and inflammatory responses. In this article we discuss the activating and inhibitory properties of the individual macrophage FcgammaR and the conditions under which these heterologous responses can occur.

To determine the role of IL-10 in cutaneous leishmaniasis, we examined lesion development following Leishmania major infection of genetically susceptible BALB/c mice lacking IL-10. Whereas normal BALB/c mice developed progressive nonhealing lesions with numerous parasites within them, IL-10(-/-) BALB/c mice controlled disease progression, and had relatively small lesions with 1000-fold fewer parasites within them by the fifth week of infection. We also examined a mechanism whereby Leishmania induced the production of IL-10 from macrophages. We show that surface IgG on Leishmania amastigotes allows them to ligate Fc gamma receptors on inflammatory macrophages to preferentially induce the production of high amounts of IL-10. The IL-10 produced by infected macrophages prevented macrophage activation and diminished their production of IL-12 and TNF-alpha. In vitro survival assays confirmed the importance of IL-10 in preventing parasite killing by activated macrophages. Pretreatment of monolayers with either rIL-10 or supernatants from amastigote-infected macrophages resulted in a dramatic enhancement in parasite intracellular survival. These studies indicate that amastigotes of Leishmania use an unusual and unexpected virulence factor, host IgG. This IgG allows amastigotes to exploit the antiinflammatory effects of Fc gamma R ligation to induce the production of IL-10, which renders macrophages refractory to the activating effects of IFN-gamma.

In this study we investigated the capacity of morphine to modulate expression of cytokines in peritoneal macrophages. Mice were implanted subcutaneously with a 75-mg morphine slow-release pellet, and 48 h later resident peritoneal macrophages were harvested. Control groups received placebo pellets, naltrexone pellets, or morphine plus naltrexone pellets. Adherent cells were stimulated with lipopolysaccharide (LPS: 10 microg/mL) plus interferon-gamma (IFN-gamma: 100 units/mL) to induce cytokine production. After 24 h RNA was extracted for analysis of cytokine mRNA levels by reverse transcriptase-polymerase chain reaction, or supernatants were collected after 48 h for determination of cytokine production by enzyme-linked immunosorbent assay (ELISA). Morphine enhanced mRNA expression of interleukin (IL)-12 p40 and tumor necrosis factor alpha (TNF-alpha) compared with controls, whereas IL-10 levels were unchanged by drug treatment. ELISA data showed that both IL-12 p40 and p70 were increased by morphine. The enhancement of IL-12 at both the mRNA and protein levels was antagonized by naltrexone, indicating that the modulation of this cytokine by morphine is via a classic opioid receptor. These results are particularly interesting in light of our previous observation that 48 h after morphine pellet implantation, the peritoneal cavity is colonized with gram-negative and other enteric bacteria. The enhancement of IL-12 by morphine might be related to morphine-induced sepsis.

In leishmaniasis, macrophages play important but potentially divergent roles. They act as the host cell in which the parasite may reside and replicate, and, at the same time, they act as an effector cell with the potential to eliminate the parasite. In this work, we experimentally induced an inflammatory model that provokes a continued recruitment of the monocytes to the site of inflammation. This model was carried out by means of implanting paraffin tablets under the skin of Balb/c or C57BL/6 mice. Mice were then infected with Leishmania major to determine how the monocyte inflammatory response to paraffin could influence the course of infection with L. major. Mice were sacrificed 15, 21, 30, and 45 days after infection, and skin and inflammatory capsule were collected for histopathology. At 15 days and 21 days, the lesions induced by L. major in combination with paraffin contained markedly increased numbers of parasites relative to lesions in parallel control animals infected with L. major (without paraffin). Both Balb/c and C57BL/6 mice exhibited high parasite numbers in their lesions. The intense parasite burden observed following paraffin implantation would suggest that the monocytes-macrophages that are recruited to the lesion are acting more as a host cell permitting parasite growth than as an effector cell capable of eliminating L. major. At later times, the two strains of mice stratified according to their genetic susceptibility/resistance profiles. Susceptible Balb/c mice continue to have large parasite burdens, whereas the resistant C56BL/6 mice begin to control parasite numbers. This later observation indicates that the genetic difference between susceptible and resistant strains is not due to differences in monocyte recruitment and cannot be reversed through the altering of monocyte inflammation.

High molecular weight kininogen (HK) and its cleaved form (HKa) have been shown to bind to neutrophils. Based on studies using monoclonal antibodies (mAbs), we postulated that CD11b/CD18 (Mac-1) might be the receptor on the neutrophils for binding to HK/HKa. However, the direct interaction of HK/HKa and Mac-1 had not been demonstrated. We therefore transfected HEK 293 cells with human Mac-1. Cell binding assays using fluorescein isothiocyanate-labeled HKa showed increased binding to the Mac-1 transfected cells compared with the control transfected cells. The binding was specific because unlabeled HKa, Mac-1-specific antibody, and fibrinogen can inhibit the binding of biotin-HKa to Mac-1 transfected cells. HKa bound to Mac-1 transfected cells (20 000 molecules/cell) with a K(d) = 62 nmol/L. To demonstrate directly the formation of a complex between HKa and Mac-1, we examined the interaction of HKa and purified Mac-1 in a cell-free system using an IAsys resonant mirror optical biosensor. The association and dissociation rate constants (k(on) and k(off), respectively) were determined, and they yielded a dissociation constant (K(d)) of 3.2x10(-9) mol/L. The functional significance of direct interaction of HKa to Mac-1 was investigated by examining the effect of HKa on cellular adhesion to fibrinogen and intercellular adhesion molecule-1 (ICAM-1), molecules abundant in the injured vessel wall. HKa blocked the adhesion of Mac-1 transfected cells to fibrinogen and ICAM-1 in a dose-dependent manner. Thus, HKa may interrupt Mac-1-mediated cell-extracellular matrix and cell-cell adhesive interactions and may therefore influence the recruitment of circulating neutrophils/monocytes to sites of vessel injury. (Blood. 2000;95:3788-3795)

Rhodococcus equi is a facultative intracellular bacterium of macrophages which can infect immunocompromised humans and young horses. In the present study, we examine the mechanism of host defense against R. equi by using a murine model. We show that bacterial killing is dependent upon the presence of gamma interferon (IFN-gamma), which activates macrophages to produce reactive nitrogen and oxygen intermediates. These two radicals combine to form peroxynitrite (ONOO(-)), which kills R. equi. Mice deficient in the production of either the high-output nitric oxide pathway (iNOS(-/-)) or the oxidative burst (gp91(phox-/-)) are more susceptible to lethal R. equi infection and display higher bacterial burdens in their livers, spleens, and lungs than wild-type mice. These in vivo observations, which implicate both nitric oxide (NO) and superoxide (O(2)(-)) in bacterial killing, were reexamined in cell-free radical-generating assays. In these assays, R. equi remains fully viable following prolonged exposure to high concentrations of either nitric oxide or superoxide, indicating that neither compound is sufficient to mediate bacterial killing. In contrast, brief exposure of bacteria to ONOO(-) efficiently kills virulent R. equi. The intracellular killing of bacteria in vitro by activated macrophages correlated with the production of ONOO(-) in situ. Inhibition of nitric oxide production by activated macrophages by using N(G)-monomethyl-L-arginine blocks their production of ONOO(-) and weakens their ability to control rhodococcal replication. These studies indicate that peroxynitrite mediates the intracellular killing of R. equi by IFN-gamma-activated macrophages.

Leishmania are intracellular protozoan parasites of macrophages. At the cellular level, the disease leishmaniasis involves the invasion of tissue macrophages by the parasite, the avoidance of cellular killing mechanisms, and the subsequent intracellular replication of parasites, with the eventual spread of the organisms to adjacent macrophages. This paper describes the process by which Leishmania organisms invade macrophages, with an overview of some of the molecules involved in this process; the mechanisms available to macrophages that have the potential to restrict the growth of Leishmania within them; and the ways that Leishmania and Leishmania-derived molecules can modulate macrophage functions and circumvent leukocyte antimicrobial responses.

The most abundant protein on the surface of the promastigote form of the protozoan parasites Leishmania spp. is a 63-kDa molecule, designated gp63 or leishmanolysin. Because gp63 has been shown to possess fibronectin-like properties, we examined the interaction of gp63 with the cellular receptors for fibronectin. We measured the direct binding of Leishmania to human macrophages or to transfected mammalian cells expressing human fibronectin receptors. Leishmania expressing gp63 exhibited modest but reproducible adhesion to human macrophages and to transfected CHO cells expressing alpha4/beta1 fibronectin receptors. In both cases, this interaction depended on gp63 but occurred independently of the SRYD sequence of gp63, because parasites expressing gp63 with a mutated SRYD sequence bound to macrophages and alpha4/beta1 receptor-expressing cells as well as did wild-type parasites. The contribution of gp63 to parasite adhesion was more pronounced when the assays were performed in the presence of complement, suggesting that the receptors for complement and fibronectin may cooperate to mediate the efficient adhesion of parasites to macrophages. The interaction of gp63 with fibronectin receptors may also play an important role in parasite internalization by macrophages. Erythrocytes to which gp63 was cross-linked were efficiently phagocytized by macrophages, whereas control erythrocytes opsonized with complement alone bound to macrophages but remained peripherally attached to the outside of the cell. Similarly, parasites expressing wild-type gp63 were rapidly and efficiently phagocytized by resting macrophages, whereas parasites lacking gp63 were internalized more slowly. This rapid internalization of gp63-expressing parasites was dependent on the beta1 integrins, because pretreatment of macrophages with monoclonal antibodies to the beta1 integrins decreased the internalization of gp63-expressing parasites. These observations indicate that complement receptors are the primary mediators of parasite adhesion; however, maximal parasite adhesion and internalization may require the participation of the beta1 integrins, which recognize fibronectin-like molecules such as gp63 on the surface of the parasite.

Appropriately regulated immune responses depend on the controlled production of cytokines from antigen presenting cells. IL-12 synthesis is tightly regulated by several redundant mechanisms. One mechanism of IL-12 regulation involves the cross-linking of surface receptors on macrophages. This pathway may be exploited by intracellular pathogens of macrophages to inhibit IL-12 production and delay or prevent the development of cell mediated immunity.

Rhodococcus equi is a facultative intracellular pathogen of macrophages and a cause of pneumonia in young horses (foals) and immunocompromised people. Isolates of R. equi from pneumonic foals typically contain large, 85- or 90-kb plasmids encoding a highly immunogenic virulence-associated protein (VapA). The objective of this study was to determine the role of the 85-kb plasmid and VapA in the intracellular survival and virulence of R. equi. Clinical isolates containing the plasmid and expressing VapA efficiently replicated within mouse macrophages in vitro, while plasmid-cured derivatives of these organisms did not multiply intracellularly. An isolate harboring the large plasmid also replicated in the tissues of experimentally infected mice, whereas its plasmid-cured derivative was rapidly cleared. All foals experimentally infected with a plasmid-containing clinical isolate developed severe bronchopneumonia, whereas the foals infected with its plasmid-cured derivative remained asymptomatic and free of visible lung lesions. By day 14 postinfection, lung bacterial burdens had increased considerably in foals challenged with the plasmid-containing clinical isolate. In contrast, bacteria could no longer be cultured from the lungs of foals challenged with the isogenic plasmid-cured derivative. A recombinant, plasmid-cured derivative expressing wild-type levels of VapA failed to replicate in macrophages and remained avirulent for both mice and foals. These results show that the 85-kb plasmid of R. equi is essential for intracellular replication within macrophages and for development of disease in the native host, the foal. However, expression of VapA alone is not sufficient to restore the virulence phenotype.

Interleukin (IL)-12 is a cytokine that possesses both proinflammatory and immunoregulatory activity. IL-12, and the interferon-gamma (IFN-gamma) that is induced by IL-12, play central roles in the development of the Th1-type immune responses that are required for immunity to intracellular pathogens. Recently a number of these pathogens, including Leishmania, measles virus, and human immunodeficiency virus (HIV), have been shown to subvert the development of cell-mediated immunity by actively inhibiting the production of IL-12. Similarly, the ligation of phagocytic receptors on macrophages has also been shown to suppress IL-12 production. The suppression of IL-12 production by receptor ligation occurs by at least two distinct mechanisms: one involves a direct inhibition of gene transcription and the other depends on the production of inhibitory cytokines. We review studies in which IL-12 has been experimentally manipulated, and we compare the mechanisms by which this regulation can occur. Because the IL-12 that is produced during acute inflammation and chronic autoimmune disorders can lead to exacerbated disease, the development of pharmacological means to suppress IL-12 production is currently under investigation. This review focuses on the production of IL-12 by antigen-presenting cells and the methods by which the down-regulation of IL-12 production can be exploited either by pathogens or for therapeutic ends.

Macrophages can respond to a variety of infectious and/or inflammatory stimuli by secreting an array of proinflammatory cytokines, the overproduction of which can result in shock or even death. In this report, we demonstrate that ligation of macrophage Fcgamma receptors (FcgammaR) can lead to a reversal of macrophage proinflammatory responses by inducing an upregulation of interleukin (IL)-10, with a reciprocal inhibition of IL-12 production. IL-10 upregulation was specific to FcgammaR ligation, since the ligation of the Mac-1 receptor did not alter IL-10 production. The identification of the specific FcgammaR subtype responsible for IL-10 upregulation was determined in gene knockout mice. Macrophages from mice lacking the FcR gamma chain, which is required for assembly and signaling by FcgammaRI and FcgammaRIII, failed to upregulate IL-10 in response to immune complexes. However, mice lacking either the FcgammaRII or the FcgammaRIII were fully capable of upregulating IL-10 production, implicating FcgammaRI in this process. The biological consequences of FcgammaRI ligation were determined in both in vitro and in vivo models of inflammation and sepsis. In all of the models tested, the ligation of FcgammaR promoted the production of IL-10 and inhibited the secretion of IL-12. This reciprocal alteration in the pattern of macrophage cytokine production illustrates a potentially important role for FcgammaR-mediated clearance in suppressing macrophage proinflammatory responses.

In the present study, we examine the cell biology of Leishmania amastigote uptake by mammalian cells and compare this process to the phagocytosis of IgG-opsonized erythrocytes. We report that many aspects of amastigote uptake into macrophages resemble classical receptor-mediated phagocytosis. Parasite uptake requires energy expenditure by macrophages but not by parasites. Treating macrophages to prevent either energy metabolism or actin polymerization prevents amastigote uptake. The uptake of amastigotes by macrophages involves the colocalization of f-actin, paxillin, and talin to phagocytic cups that are formed around amastigotes during internalization. Treatment of macrophages with genestein, to inhibit protein phosphorylation, prevents amastigote uptake, indicating that this process, like receptor-mediated phagocytosis, depends on protein tyrosine phosphorylation. However, the amount and the pattern of protein tyrosine phosphorylation observed during amastigote uptake by macrophages is reduced relative to that observed during IgG-erythrocyte phagocytosis. The uptake of viable, but not heat-killed amastigotes, is associated with a decrease in the intensity of several specific macrophage proteins that are phosphorylated on tyrosine residues. In summary, although many features of amastigote uptake by macrophages resemble classical receptor-mediated phagocytosis, differences in macrophage protein phosphorylation during amastigote phagocytosis may contribute to the unique aspects of amastigote uptake and intracellular survival in macrophages.

Interleukin (IL)-12 is a monocyte- and macrophage-derived cytokine that plays a crucial role in both the innate and the acquired immune response. In this study, we examined the effects that ligating specific macrophage receptors had on the induction of IL-12 by lipopolysaccharide (LPS). We report that ligation of the macrophage Fcgamma, complement, or scavenger receptors inhibited the induction of IL-12 by LPS. Both mRNA synthesis and protein secretion were diminished to near-undetectable levels following receptor ligation. Suppression was specific to IL-12 since IL-10 and tumor necrosis factor-alpha (TNF-alpha) production were not inhibited by ligating macrophage receptors. The results of several different experimental approaches suggest that IL-12 downregulation was due to extracellular calcium influxes that resulted from receptor ligation. First, preventing extracellular calcium influxes, by performing the assays in EGTA, abrogated FcgammaR-mediated IL-12(p40) mRNA suppression. Second, exposure of macrophages to the calcium ionophores, ionomycin or A23187, mimicked receptor ligation and inhibited IL-12(p40) mRNA induction by LPS. Finally, bone marrow-derived macrophages from FcR gamma chain-deficient mice, which fail to flux calcium after receptor ligation, failed to inhibit IL-12(p40) mRNA induction. These results indicate that the calcium influxes that occur as a result of receptor ligation are responsible for inhibiting the induction of IL-12 by LPS. Hence, the ligation of phagocytic receptors on macrophages can lead to a dramatic decrease in IL-12 induction. This downregulation may be a way of limiting proinflammatory responses of macrophages to extracellular pathogens, or suppressing the development of cell-mediated immunity to intracellular pathogens.

Leishmania are intracellular protozoan parasites which reside primarily, if not exclusively, in host mononuclear phagocytes. Several studies have demonstrated that infectious promastigotes rapidly and efficiently fix complement when they encounter serum components during their transmission to the mammalian host. Activation of the complement system by a microorganism can have 3 distinct biological effects. First, fixation of the terminal complement components can result in complement-mediated lysis. Second, fixation of the 3rd component of complement can lead to opsonization of the organism for uptake by phagocytic cells. Finally, the elaboration of the complement anaphylotoxins, C3a and C5a, can lead to inflammation. In the present chapter, we discuss the interaction of leishmania promastigotes with the complement system. We show that infectious promastigotes avoid the lytic effects of complement and resist fixation of the terminal complement components. At the same time, however, these organisms depend on fixation of opsonic complement to invade host mononuclear phagocytes efficiently. We discuss the mechanisms which allow metacyclic leishmania promastigotes to exploit the opsonic properties of complement and the receptors on macrophages involved in leishmania recognition. The role of complement mediated inflammatory processes in the host response to leishmania infection is an area which requires additional study.

Amplification of the Cap b locus of Haemophilus influenzae occurs frequently in clinical isolates and has been proposed to be a mechanism by which this organism evades host defense. To determine if amplification of this locus affected complement fixation, in vitro studies to determine complement-mediated bacteriolysis and complement-mediated opsonization of an isogenic set of organisms containing 2, 3, and 4 copies of the Cap b locus were performed. Organisms containing 4 copies of the Cap b locus were significantly more resistant to antibody-dependent, classical complement pathway-directed bacteriolysis than were organisms containing 2 copies. Organisms containing 3 copies of this locus exhibited intermediate susceptibility to lysis. Complement-mediated opsonization of these organisms was assessed by determining the degree of binding of bacteria to murine or human macrophages or to nonphagocytic cells transfected with the genes for human Mac-1, the complement receptor type 3. In all three assay systems, organisms containing 4 copies of the Cap b locus bound less well than did organisms containing 2 copies of this locus. Consistent with their decreased susceptibility to lysis and opsonization, organisms with 4 copies of the Cap b locus fixed less C3 than did organisms containing 2 copies. These data demonstrate that amplification of the Cap b locus is associated with decreased susceptibility to complement-mediated lysis and decreased complement-mediated opsonization and suggest that amplification is used by these pathogens to increase their resistance to complement-dependent host defense mechanisms [correction of mecanisms].

Complement has long been acknowledged as an important component of host defense to extracellular pathogens. David Mosser and Andrew Brittingham here describe how Leishmania spp, intracellular pathogens of mononuclear phagocytes, exploit the opsonic and chemotactic effects of complement to initiate infection in the mammalian host.

Two disintegrins with a high degree of amino acid sequence similarity, echistatin and eristostatin, showed a low level of interaction with Chinese hamster ovary (CHO) cells, but they bound to CHO cells transfected with alpha IIb beta 3 genes (A5 cells) and to CHO cells transfected with alpha v beta 3 genes (VNRC3 cells) in a reversible and saturable manner. Scatchard analysis revealed that eristostatin bound to 816000 sites per A5 cell (Kd 28 nM) and to 200000 sites (Kd 14 nM) per VNRC3 cell respectively. However, VNRC3 cells did not bind to immobilized eristostatin. Echistatin bound to 495000 sites (Kd 53 nM) per A5 cell and to 443000 sites (Kd 20 nM) per VNRC3 cell. As determined by flow cytometry, radiobinding assay and adhesion studies, binding of both disintegrins to A5 cells and resting platelets and binding of echistatin to VNRC3 cells resulted in the expression of ligand-induced binding sites (LIBS) on the beta 3 subunit. Eristostatin inhibited, more strongly than echistatin, the binding of three monoclonal antibodies: OPG2 (RGD motif dependent), A2A9 (alpha IIb beta 3 complex dependent) and 7E3 (alpha IIb beta 3 and alpha v beta 3 complex dependent) to A5 cells, to resting and to activated platelets and to purified alpha IIb beta 3. Experiments in which echistatin and eristostatin were used alone or in combination to inhibit the binding of 7E3 and OPG2 antibodies to resting platelets suggested that these two disintegrins bind to different but overlapping sites on alpha IIb beta 3 integrin. Monoclonal antibody LM 609 and echistatin seemed to bind to different sites on alpha v beta 3 integrin. However, echistatin inhibited binding of 7E3 antibody to VNRC3 cells and to purified alpha v beta 3 suggesting that alpha v beta 3 and alpha IIb beta 3 might share the same epitope to which both echistatin and 7E3 bind. Eristostatin had no effect in these systems, providing further evidence that it binds to a different epitope on alpha v beta 3.

We analyzed the binding of sheep erythrocytes bearing C3b (EC3b) to cells transfected with human complement receptors. EC3b bound avidly to cells expressing CR1 but failed to bind to cells expressing CR3. In the presence of factor I, the binding of EC3b, to CR1 was transient. Primary monocytes and cotransfected cells expressing both CR1 and CR3 mediated a stable resetting of EC3b, even in the prolonged presence of factor I. This stable adhesion was dependent on the presence of CR3, because blocking CR3 with mAb resulted in the factor I-dependent release of erythrocytes from these cells. A model is proposed in which these two complement receptors cooperate in a unique manner. These results suggest that the stable adhesion of complement-opsonized particles to cells expressing CR1 and CR3 is actually a dynamic molecular process in which an important function of leukocyte CR1 is to generate the ligands for CR3.

It has been suggested that the developmental maturation of Leishmania major promastigotes can affect their interaction with human complement receptors. To study this, we measured the adhesion of metacyclic and logarithmic-phase L. major promastigotes to complement receptors expressed on primary macrophages, to recombinant receptors expressed on transfected cells, or to purified complement receptors in a cell-free system. We demonstrate that complement-opsonized promastigotes can bind to both Mac-1 and complement receptor type 1 (CR1) and that the transition of promastigotes from the noninfectious logarithmic phase of growth to the infectious metacyclic stage does not affect this interaction. Furthermore, we show that Mac-1 and CR1 can cooperate to mediate the efficient adhesion of complement-opsonized metacyclic promastigotes to cells expressing both receptors. On human monocyte-derived macrophages, Mac-1 appears to make a quantitatively greater contribution to this adhesion than does CR1, since blocking macrophage Mac-1 diminishes metacyclic promastigote adhesion to a greater extent than does blocking CR1. In addition, bovine monocytes lacking Mac-1 exhibit a dramatic decrease in complement-dependent promastigote adhesion, relative to normal monocytes. The predominance of Mac-1 in these interactions is due, at least in part, to the factor I cofactor activity of CR1, which facilitates the conversion of C3b to iC3b. The stable adhesion of complement-opsonized metacyclic promastigotes to Mac-1 is a prerequisite for phagocytosis by human monocyte-derived macrophages. Blocking Mac-1 on macrophages abrogates the majority of the complement-dependent phagocytosis of promastigotes, whereas blocking CR1 has no detectable effect on phagocytosis. In addition, bovine monocytes lacking Mac-1 exhibit a dramatic reduction in promastigote phagocytosis relative to normal bovine monocytes. We conclude, therefore, that the two complement receptors, Mac-1 and CR1, can cooperate to mediate the initial complement-dependent adhesion of metacyclic promastigotes to human monocyte-derived macrophages and that Mac-1 is the predominant complement receptor responsible for the phagocytosis of complement-opsonized metacyclic promastigotes.

Rhodococcus equi is emerging as a cause of pneumonia in immunocompromised people, especially those with AIDS. Like mycobacteria, R. equi is phagocytosed by alveolar macrophages and replicates within them. Recent work is beginning to elucidate the cell and molecular biology of this opportunistic pathogen and the host immune response to it.

Ethylenediamine extraction of the cell walls of Candida albicans yields an extract which possesses antigenic activity, and like other whole cell and cell wall preparations of Candida, has been shown previously to possess immunomodulatory activity. We report here results which show that non-elicited, resident peritoneal macrophages treated with the ethylenediamine cell wall extract exhibit morphological characteristics of activated macrophages. Treated cells also manifest an increased respiratory burst response and secrete elevated levels of IL-6. The treatment with the extract appears to provide a co-priming signal to the resting macrophages, since co-stimulation with interferon-gamma results in an increased level of IL-6 and IL-1. Our results also show that extract-treated cells manifest increased responsiveness to a lipopolysaccharide as measured by the production of tumour necrosis factor-alpha. These results provide an additional basis for our understanding of the immunomodulatory activity of components of the Candida cell wall.

The Leishmania surface protease gp63 has been identified as a parasite virulence factor. To better define the role of gp63 in Leishmania infectivity, the interaction of recombinant gp63 with complement and complement receptors was examined. On Leishmania, gp63 was not necessary for complement fixation. Complement activation occurred on transfected organisms expressing varying amounts of gp63 and on organisms expressing a mutant form of gp63 devoid of proteolytic activity. However, organisms expressing wild-type gp63 on their surface fixed only small amounts of the terminal complement components and were dramatically more resistant to lysis by complement than were those lacking functional gp63. Organisms expressing wild-type gp63 more rapidly converted C3b on their surface to a form that exhibited the neoantigen of iC3b and interacted avidly with cells expressing Mac-1, the receptor for iC3b. Complement fixation by transfected mammalian cells expressing recombinant Leishmania gp63 on their surface was also examined. The presence of Leishmania gp63 on the surface of these cells converted them into efficient activators of complement. Cells expressing gp63 on their surface fixed complement and bound avidly to the human complement receptors. The proteolytic activity of this molecule was not necessary for complement activation or adhesion to complement receptors. Thus, gp63 may contribute to parasite virulence by exerting a novel type of control over complement fixation. Organisms expressing gp63 can exploit the opsonic properties of complement while avoiding its lytic effects.

Candida albicans exhibits hemolytic activity when grown on glucose-enriched blood agar. This activity is present on intact organisms, and it is secreted into the culture medium. Hemoglobin released from lysed erythrocytes can restore the transferrin-inhibited growth of C. albicans. We conclude that C. albicans expresses a hemolytic factor which allows it to acquire iron from host erythrocytes.