RESUMEN
After entering a cell and undergoing reverse transcription, the retroviral genome is contained in a preintegration complex (PIC) that mediates its integration into host cell DNA. PICs have been shown to prefer torsionally strained DNA, but the effect of target DNA length has not been previously examined. In this report, concatemerization of a repeating 105-base pair unit was used to vary target DNA length independently from basic DNA sequence, while maintaining both PICs and target DNAs in solution. Integration junctions were quantified by real-time fluorescence-monitored polymerase chain reaction amplification using primers in the viral long terminal repeat and the target DNA. Unreacted target DNA severely inhibited the post-reaction polymerase chain reaction detection step, requiring its removal using lambda exonuclease digestion. Integration into a 32-unit concatemer of target DNA was markedly more efficient than integration into a monomeric unit, indicating that longer target DNA was preferred. This substrate was used to construct a simple, robust, and adaptable assay that can serve as a method for studying the host cell factors that enhance PIC integration, and as a drug discovery platform for integration inhibitors active against PICs.
Asunto(s)
ADN Viral/genética , VIH-1/genética , VIH-1/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Unión Competitiva , Citoplasma/metabolismo , ADN/metabolismo , Cartilla de ADN/metabolismo , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Modelos Genéticos , Plásmidos/metabolismo , Unión Proteica , Sales (Química)/farmacología , Especificidad por Sustrato , Factores de TiempoRESUMEN
Bacterial DNA and its synthetic immunostimulatory oligodeoxynucleotide analogs (ISS-ODN) activate innate immunity and promote Th1 and cytotoxic T-lymphocyte immune responses. Based on these activities, we investigated whether ISS-ODN could modify the course of Mycobacterium avium infection. M. avium growth in vitro was significantly inhibited by ISS-ODN treatment of human and mouse macrophages, and M. avium growth in vivo was similarly inhibited in C57BL/6 mice treated with ISS-ODN. This protective effect of ISS-ODN was largely independent of tumor necrosis factor alpha (TNF-alpha), interleukin 12 (IL-12), nitric oxide, NADPH oxidase, alpha/beta interferon (IFN-alpha/beta), and IFN-gamma. In contrast, we found that the induction of indoleamine 2,3-dioxygenase (IDO) was required for the antimycobacterial effect of ISS-ODN. To evaluate the potential for synergism between ISS-ODN and other antimycobacterial agents, treatment with a combination of ISS-ODN and clarithromycin (CLA) was tested in vitro and in vivo. ISS-ODN significantly enhanced the therapeutic effect of CLA in both human and mouse macrophages and in C57BL/6 mice. This study newly identifies IDO as being involved in the antimicrobial activity of ISS-ODN and suggests the usefulness of ISS-ODN when used in combination with conventional chemotherapy for microbial infections.
Asunto(s)
Adyuvantes Inmunológicos , Oligodesoxirribonucleótidos/inmunología , Tionucleótidos/inmunología , Triptófano Oxigenasa/inmunología , Tuberculosis/inmunología , Animales , Antibacterianos/uso terapéutico , Células Cultivadas , Claritromicina/farmacología , ADN/inmunología , ADN/uso terapéutico , Modelos Animales de Enfermedad , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa , Interferón-alfa/inmunología , Interferón beta/inmunología , Interferón gamma/inmunología , Interleucina-12/inmunología , Macrófagos/citología , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Ratones Noqueados , Monocitos/citología , Monocitos/inmunología , Monocitos/microbiología , Mycobacterium avium/crecimiento & desarrollo , Mycobacterium avium/inmunología , NADPH Oxidasas/inmunología , Óxido Nítrico Sintasa/inmunología , Óxido Nítrico Sintasa de Tipo II , Oligodesoxirribonucleótidos/uso terapéutico , Linfocitos T/inmunología , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiología , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
CD40 ligand (also called CD40L, CD154, or TNFSF5) is a membrane protein expressed mainly by activated CD4+ T cells, which interacts with its receptor, CD40, on a variety of cells. The crucial importance of the CD40L-CD40 system for many immune responses has been extensively described. This review focuses on the multiple roles that this system may play in HIV infection. In early HIV infection, CD40L expression contributes to the immunological control of viral replication by inducing HIV-suppressive chemokines and supporting the production of anti-HIV antibodies and cytotoxic T cells. However, by activating antigen-presenting cells, such as dendritic cells and macrophages, CD40L can also lead to increased CD4+ T cell activation, which promotes the replication of HIV in these lymphocytes. Later, with the development of AIDS, CD40L-expressing CD4+ T cells become selectively depleted, perhaps as a result of a gp120-induced signal through CD4 that down-regulates CD40L expression. This acquired CD40L deficiency may explain the similarity between the types of opportunistic infections that occur in AIDS and in congenital CD40L deficiency. Vaccines or other strategies that promote the growth of CD4+ T cells capable of expressing CD40L may help to sustain host immunity against HIV and prevent AIDS-defining opportunistic infections.
Asunto(s)
Infecciones por VIH/inmunología , VIH/inmunología , Glicoproteínas de Membrana/inmunología , Animales , Ligando de CD40 , HumanosRESUMEN
Bacterial genomic DNA, plasmid DNA (pDNA) and synthetic oligodeoxynucleotides (ODN) containing immunostimulatory DNA sequences (ISS) have been proposed to foster a Th1 response via the release of type 1 cytokines from macrophages, dendritic cells, NK cells and B cells. In this study, we show that ISS-enriched DNA up-regulates a distinct profile of cell surface molecules on macrophages and B cells in vitro and in vivo. ISS-ODN and ISS-containing pDNA enhanced the expression of antigen presentation molecules (MHC class I and II), co-stimulatory molecules (B7-1, B7-2 and CD40), cytokine receptors (IFN-gamma receptor and IL-2 receptor), an adhesion molecule (ICAM-1) and an Fc receptor (Fcgamma receptor) on murine B cells or bone marrow-derived macrophages. The increased expression of these surface molecules is seen in purified cell populations and is largely independent of the effects of type 1 cytokines. Splenic antigen-presenting cells stimulated with ISS-ODN in vivo efficiently activate naive T cells and bias their differentiation toward a Th1 phenotype in vitro. Thus, the induction of both type 1 cytokines and a distinct profile of cell surface molecules contributes to the potent immunostimulatory effects of ISS-containing DNA on innate and adaptive immunity.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , ADN Bacteriano/inmunología , ADN Viral/inmunología , Proteínas de la Membrana/inmunología , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Células de la Médula Ósea/inmunología , ADN Bacteriano/genética , ADN Bacteriano/farmacología , ADN Viral/genética , ADN Viral/farmacología , Femenino , Activación de Linfocitos/inmunología , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Oligonucleótidos/inmunología , Oligonucleótidos/farmacología , Bazo/citología , Linfocitos T/inmunología , Células TH1/inmunología , Regulación hacia ArribaRESUMEN
Mycobacterium avium is a common opportunistic pathogen in immunocompromised patients such as those infected with human immunodeficiency virus. Although M. avium is an intracellular organism replicating predominantly in macrophages, disseminated M. avium infection is seen in AIDS patients with CD4(+) cell counts of <50 cells/microliters, suggesting a possible involvement of a T cell-macrophage interaction for the elimination of M. avium. To determine whether CD40-CD40 ligand (CD40L) interactions play a role in M. avium infection, we studied the ability of CD40L to restrict M. avium replication in human monocyte-derived macrophages (MDM) in vitro. MDM were infected with M. avium and cocultured with CD40L-transfected 293 cells for 7 days. Intracellular growth of M. avium in these MDM was assessed by colony counting. CD40L-expressing cells inhibited growth of M. avium in MDM by 86.5% +/- 4.2% compared to MDM cultured with control cells. These findings were verified by assays using purified, soluble recombinant human CD40L (CD40LT). CD40LT (5 micrograms/ml) inhibited intracellular growth of M. avium by 76.9% +/- 18.0% compared to cells treated with medium alone. Inhibition by CD40LT was reduced by monoclonal antibodies (MAbs) against CD40 and CD40L. The inhibitory effect of CD40LT was not accompanied by enhancement of interleukin-12 (IL-12) production by M. avium-infected MDM, while CD40L-expressing cells stimulated IL-12 production by these cells. Treatment of M. avium-infected mice with MAb against murine CD40L resulted in recovery of larger numbers of organisms (0.8 to 1.0 log) from the spleens, livers, and lungs of these animals compared to infected mice which received normal immunoglobulin G. These results indicate that CD40-CD40L signaling may be an important step in host immune response against M. avium infection.
Asunto(s)
Antígenos CD40/inmunología , Macrófagos/inmunología , Glicoproteínas de Membrana/inmunología , Mycobacterium avium/inmunología , Linfocitos T/inmunología , Tuberculosis/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Antígenos de Diferenciación de Linfocitos T/inmunología , Ligando de CD40 , Humanos , Inmunidad Celular , Ligandos , Macrófagos/microbiología , Ratones , Proteínas Recombinantes/inmunología , Tuberculosis/tratamiento farmacológicoRESUMEN
beta-chemokines play an important role in the development of immunologic reactions. Macrophages are major beta-chemokine-producing cells during T-cell directed, delayed-type hypersensitivity reactions in tissues, and have been reported to be important producers of beta-chemokines in the lymph nodes of HIV-1-infected individuals. However, the physiological signals responsible for inducing macrophages to produce beta-chemokines have not been established. Two soluble T cell products, interferon-gamma and granulocyte-macrophage colony stimulating factor, were added to cultured macrophages, but failed to stimulate the production of macrophage inflammatory protein-1alpha and -1beta; regulated upon activation, normal T cell expressed and secreted (RANTES); or monocyte chemoattractant protein-1. Instead, direct cell-cell contact between macrophages and cells engineered to express CD40L (also known as CD154) resulted in the production of large amounts of macrophage inflammatory protein-1alpha and -1beta, and RANTES (all ligands for CCR5), and monocyte chemoattractant protein-1 (a ligand for CCR2). Supernatants from CD40L-stimulated macrophages protected CD4(+) T cells from infection by a nonsyncytium-inducing strain of HIV-1 (which uses CCR5 as a coreceptor). These results have implications for granulomatous diseases, and conditions such as atherosclerosis and multiple sclerosis, where CD40L-bearing cells have been found in the macrophage-rich lesions where beta-chemokines are being produced. Overall, these findings define a pathway linking the specific recognition of antigen by T cells to the production of beta-chemokines by macrophages. This pathway may play a role in anti-HIV-1 immunity and the development of immunologic reactions or lesions.
Asunto(s)
Quimiocinas CC/biosíntesis , VIH-1/crecimiento & desarrollo , Macrófagos/fisiología , Glicoproteínas de Membrana/fisiología , Ligando de CD40 , Comunicación Celular , Membrana Celular/fisiología , Células Cultivadas , Quimiocina CCL2/biosíntesis , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Interferón gamma/farmacología , Linfocitos T/inmunología , Linfocitos T/virología , Replicación ViralRESUMEN
An adjuvant role for certain short bacterial immunostimulatory DNA sequences (ISSs) has recently been proposed on the basis of their ability to stimulate T helper-1 (Th1) responses in gene-vaccinated animals. We report here that noncoding, ISS-enriched plasmid DNAs or ISS oligonucleotides (ISS-ODNs) potently stimulate immune responses to coadministered antigens. The ISS-DNAs suppress IgE synthesis, but promote IgG and interferon-gamma (IFN-gamma) production. They furthermore initiate the production of IFN-gamma, IFN-alpha, IFN-beta, and interleukins 12 and 18, all of which foster Th1 responses and enhance cell-mediated immunity. Consideration should be given to adding noncoding DNA adjuvants to inactivated or subunit viral vaccines that, by themselves, provide only partial protection from infection.
Asunto(s)
Adyuvantes Inmunológicos , ADN/inmunología , Activación de Linfocitos/genética , Células TH1/inmunología , Animales , Formación de Anticuerpos/genética , ADN/genética , Femenino , Inmunoglobulina E/biosíntesis , Inmunoglobulina G/biosíntesis , Interferones/biosíntesis , Interleucinas/biosíntesis , Activación de Macrófagos/genética , Ratones , Ratones Endogámicos BALB C , beta-Galactosidasa/inmunologíaAsunto(s)
Infecciones por VIH/inmunología , VIH-1/fisiología , Macrófagos/inmunología , Linfocitos T/inmunología , Síndrome de Inmunodeficiencia Adquirida/inmunología , Linfocitos T CD4-Positivos/inmunología , Citocinas/fisiología , Humanos , Macrófagos/virología , Proteínas de la Membrana/fisiología , Receptores CXCR4 , Receptores del VIH/fisiología , Replicación ViralRESUMEN
HIV-related immune deficiency differs from that due to other causes in its severity and inexorably progressive nature. It leads to multiple opportunistic infections in patients with advanced HIV infection, mostly with common endogenous and environmental organisms that usually pose little threat to human health.
Asunto(s)
Infecciones Oportunistas Relacionadas con el SIDA/inmunología , Infecciones por VIH/inmunología , Infecciones Oportunistas Relacionadas con el SIDA/fisiopatología , Anticuerpos Antivirales/inmunología , Linfocitos B/inmunología , Infecciones por VIH/fisiopatología , Humanos , Inmunidad CelularRESUMEN
Dextran sulfate is a potent inhibitor of human immunodeficiency virus (HIV) binding and replication in lymphocytic cell lines. In this study, we demonstrate that the effect of dextran sulfate and heparin depends on the host cell type and on the V3 loop, the principal neutralizing determinant of HIV gp120. In particular, when dextran sulfate was tested on primary human macrophages infected with macrophage-tropic viruses, enhancement of infection was observed in 6 of 11 independent macrophage preparations and with 5 of 13 primary HIV isolates. Our in vitro observations might explain why enhanced HIV replication was observed in HIV-infected patients treated with dextran sulfate.
Asunto(s)
Sulfato de Dextran/farmacología , Proteína gp120 de Envoltorio del VIH/fisiología , VIH-1/efectos de los fármacos , Heparina/farmacología , Línea Celular , Humanos , Replicación Viral/efectos de los fármacosRESUMEN
Apoptosis is a major cause of cell death in health and disease. In contrast to necrosis, apoptosis does not induce an inflammatory response and the cellular debris produced by apoptosis has been assumed to be biologically inert. This review challenges this assumption by suggesting that apoptotic debris (especially in the context of growing tumors or during HIV infection) may have immunological activities, mainly immunosuppressive but perhaps also immunostimulatory. In many cases, the surface of apoptotic cells differs from normal cells in that phosphatidylserine (PS) is aberrantly exposed on the external face of the cell membrane. Liposomes composed of PS may down-modulate macrophage anti-leishmanial activities, suppress macrophage TNF production, suppress lymphocyte proliferation, and increase macrophage proliferation. "Membrane shedding" has been described in certain malignancies where apoptosis may be occurring, and the shed tumor membrane vesicles have been shown to reduce MHC class II expression on macrophages and decrease lymphocyte responsiveness, perhaps because of their ganglioside content. Finally, the apoptotic debris from HIV-infected cells may bear on its surface viral proteins which contain immunosuppressive peptide sequences. This debris may also use viral envelope proteins to fuse into macrophages and thereby avoid phagocytosis and lysosomal destruction. These considerations suggest that the flux of apoptosing cells and debris through the immune system that occurs during tumor growth and HIV infection should not be assumed to be immunologically neutral. In particular, HIV-related apoptosis may have immunosuppressive effects in addition to the numerical depletion of lymphocytes.
Asunto(s)
Apoptosis/inmunología , Infecciones por VIH/inmunología , Neoplasias/inmunología , Infecciones por VIH/patología , Humanos , Tolerancia Inmunológica/fisiología , Inmunosupresores/farmacología , Neoplasias/patología , Fosfatidilserinas/farmacología , Fosfatidilserinas/fisiologíaAsunto(s)
Síndrome de Inmunodeficiencia Adquirida/fisiopatología , Células Dendríticas/microbiología , Células Dendríticas/fisiología , VIH/fisiología , Macrófagos/microbiología , Macrófagos/fisiología , Síndrome de Inmunodeficiencia Adquirida/patología , Comunicación Celular/fisiología , Células Dendríticas/patología , VIH/aislamiento & purificación , Humanos , Macrófagos/patologíaRESUMEN
Apoptosis is a major form of cell death in HIV infection. This review presents current ideas on the role of apoptosis in the development of AIDS. HIV may cause apoptosis either directly in individual CD4+ T cells through cellular infection and through the release of gp120 envelope protein, or indirectly by initiating systemic disturbances in the immune system. Furthermore, although apoptosis is often assumed to be a biological dead end, linear, unintegrated retroviral DNA survives apoptosis in avian leukosis virus systems. Macrophages avidly phagocytose apoptosing cells, and the viral DNA in apoptotic debris might spontaneously transfect macrophages and lead to the production of new virions. Such a hypothetical accessory infection pathway may explain why anti-HIV cytotoxic cells are unable to clear this virus from the body. Strategies directed against the "recycling" of the retroviral genomes present in apoptotic debris may ultimately have a role in the treatment of HIV infection.
Asunto(s)
Apoptosis/fisiología , Infecciones por VIH/fisiopatología , Macrófagos/microbiología , Macrófagos/patología , Linfocitos T/microbiología , Linfocitos T/patología , Antígenos CD4/análisis , VIH/aislamiento & purificación , Infecciones por VIH/patología , Humanos , Macrófagos/fisiología , Fagocitosis/fisiología , Linfocitos T/inmunologíaRESUMEN
Lentiviruses, including human immunodeficiency virus type 1 (HIV-1), are unusual among retroviruses in their ability to infect nondividing cells. The matrix proteins of several lentiviruses contain a short stretch of amino acids reminiscent of known nuclear localization signals. In HIV-1, this motif has been shown to function as a nuclear targeting sequence when conjugated to a heterologous protein, and to permit the active nuclear import of the HIV-1 preintegration complex in growth-arrested cells. In the present work, mutations were introduced in the matrix nuclear localization region of T-cell- and macrophage-tropic HIV-1 clones. The resulting viral mutants replicated with normal or even accelerated kinetics in dividing cells, including activated peripheral blood lymphocytes. However, in sharp contrast with wild-type virus, the mutants could not grow efficiently in terminally differentiated macrophages or establish a stable and inducible infection intermediate in unstimulated peripheral blood lymphocytes. Because macrophages represent a major viral reservoir in vivo, and because at any given time most T cells in the body are quiescent, these results strongly suggest that the karyophilic properties of the matrix protein are critical for the spread of the virus in HIV-infected individuals, and consequently for AIDS pathogenesis.
Asunto(s)
Núcleo Celular/metabolismo , Productos del Gen gag/metabolismo , Antígenos VIH/metabolismo , VIH-1/fisiología , Macrófagos/microbiología , Señales de Clasificación de Proteína/metabolismo , Linfocitos T/microbiología , Proteínas Virales , División Celular , Línea Celular , Productos del Gen gag/genética , Antígenos VIH/genética , VIH-1/genética , Macrófagos/citología , Mutagénesis , Reacción en Cadena de la Polimerasa , Linfocitos T/citología , Replicación Viral , Productos del Gen gag del Virus de la Inmunodeficiencia HumanaRESUMEN
Interferons (IFNs) inhibit the replication of a wide range of animal viruses by acting at various steps of the life cycle. Interferons display a particularly potent antiviral effect on HIV-1 replication in primary human macrophages. A high virus-to-cell multiplicity of infection was used to investigate which steps in a single replicative cycle in these primary human cells were affected by IFNs. Monocyte-derived macrophages from healthy seronegative donors were infected with HIV-1BaL. Virus production was assessed by immunoassay for p24 antigen. Viral DNA was detected by PCR while mRNA was detected specifically by RT-PCR with primers bracketing the 5' introns of HIV-1 to detect only spliced transcripts such as tat, rev, nef, and env mRNAs. Macrophages pretreated with IFN-alpha, -beta or -gamma had a reduced viral DNA signal while the spliced mRNA signal was essentially abolished. No virus was produced. To test whether IFNs could reduce HIV transcripts in cells with established productive infection, macrophages were infected and reinfection was then prevented by azidothymidine before starting interferon treatment. Under such conditions, the addition of interferons did not affect significantly the levels of HIV spliced transcripts. No intracellular accumulation of p24 antigen was observed. Therefore, the major effect of IFNs was at an early step of the virus life cycle and resulted in a reduced viral DNA synthesis.
Asunto(s)
VIH/efectos de los fármacos , Interferones/farmacología , Macrófagos/microbiología , Replicación Viral/efectos de los fármacos , Células Cultivadas , ADN Viral/genética , Genes Virales/genética , VIH/genética , VIH/fisiología , Proteína p24 del Núcleo del VIH/análisis , Humanos , Interferón-alfa/farmacología , Interferón beta/farmacología , Interferón gamma/farmacología , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Viral/genética , Replicación Viral/fisiologíaRESUMEN
The prototypic macrophage-tropic HIV-1 isolate, HIV-1BaL, cannot replicate in the monocytoid cell line THP-1. After induction of differentiation by a phorbol diester, a fraction of THP-1 cells became permissive to HIV-1BaL. In contrast, this treatment decreased permissiveness for the lymphotropic isolate HIV-1LAI. Viral DNA was not synthesized in unstimulated THP-1 cells, as determined with PCR, suggesting that the block to HIV-1BaL replication in these cells occurred at an early step of the virus replicative cycle prior to or at the level of reverse transcription. Virus binding studies showed that differences in cell permissiveness for HIV-1BaL were not due to altered virus binding. Substantial amounts of HIV-1BaL bound to both undifferentiated and differentiated THP-1 cells, and this binding could not be prevented by blocking with the anti-CD4 antibody Leu3a, which did prevent the binding of HIV-1LAI to CEM T lymphoid cells. While Leu3a was very effective at preventing the infection by HIV-1LAI in CEM cells, it was less effective in preventing HIV-1BaL infection of differentiated THP-1 cells or primary macrophages. Although it is likely that molecules other than CD4 on monocytic cells can mediate binding of macrophage-tropic HIV, the binding of HIV-1BaL to THP-1 cells was not sufficient for infection, because binding was the same in nonpermissive undifferentiated cells as in permissive differentiated cells. Thus, the restriction of viral replication in this model cell system occurs at some step after virion binding. Comparison of differentiated THP-1 cells with their undifferentiated counterparts may provide an approach to defining cellular determinants of HIV host range other than CD4 expression and to characterizing the incompletely defined steps of viral entry.
Asunto(s)
VIH-1/fisiología , Macrófagos/microbiología , Monocitos/microbiología , Replicación Viral/fisiología , Anticuerpos Monoclonales , Antígenos CD4/fisiología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , ADN Viral/biosíntesis , Humanos , Modelos Biológicos , Monocitos/citología , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales CultivadasRESUMEN
We studied the effects of vitamin D3 compounds on the replication of human immunodeficiency virus type 1 (HIV-1) in the monoblastoid cell line U937 and in primary monocyte-derived macrophage cultures to understand how modulators of monocyte/macrophage effector function might affect the pathogenesis of HIV-1 infection. U937 cell cultures exposed to 1, alpha 25-dihydroxyvitamin D3 prior to HIV-1 infection showed enhanced virus replication that was apparently due to increased cellular resistance to viral cytopathic effects; a marked inhibition of virus replication was noted in cells exposed to 1 alpha,25-dihydroxyvitamin D3 subsequent to infection. Exposure of blood-derived monocyte/macrophages to vitamin D3 compounds prior to infection also affected virus growth; in most cases, substantial inhibition of HIV-1 replication was noted in vitamin D3-treated macrophage cultures. Our results demonstrate that vitamin D3 compounds with recognized abilities to induce cellular differentiation can modulate HIV-1 infection of human macrophages.