RESUMO
Macrophage-activating lipopeptide-2 (MALP-2) is a potent inducer of proinflammatory cytokine secretion by macrophages, monocytes, and dendritic cells. MALP-2 was reported to be involved in natural killer (NK) cell activation and ensuing tumor rejection. However, the mechanism of MALP-2-mediated NK cell activation remained unclear. Therefore, we studied the effects of MALP-2 on cultured human NK cells. We found that MALP-2 had no direct effect on NK cells. Instead, MALP-2 acted on monocytes and triggered the release of different molecules such as interleukin (IL)-1ß, IL-6, IL-10, IL-12, IL-15, interferon gamma-induced protein (IP-10), and prostaglandin (PG)-E2. Our data show that monocyte-derived IP-10 could significantly induce NK cell cytotoxicity as long as the immunosuppression by PGE2 is specifically inhibited by cyclooxygenase (COX)-2 blockade. In summary, our results show that MALP-2-mediated stimulation of monocytes results in the production of several mediators which, depending on the prevailing conditions, affect the activity of NK cells in various ways. Hence, MALP-2 administration with concurrent blocking of COX-2 can be considered as a promising approach in MALP-2-based adjuvant tumor therapies.
Assuntos
Inibidores de Ciclo-Oxigenase 2/farmacologia , Dinoprostona/antagonistas & inibidores , Células Matadoras Naturais/efeitos dos fármacos , Lipopeptídeos/farmacologia , Monócitos/efeitos dos fármacos , Receptor 2 Toll-Like/agonistas , Receptor 6 Toll-Like/agonistas , Células Cultivadas , Inibidores de Ciclo-Oxigenase 2/imunologia , Dinoprostona/biossíntese , Dinoprostona/imunologia , Humanos , Células Matadoras Naturais/imunologia , Lipopeptídeos/imunologia , Monócitos/imunologia , Transdução de SinaisRESUMO
Toll-like receptors (TLRs) are known primarily as pathogen recognition receptors of the innate immunity, initiating inflammatory pathways to organize the immune defense. More recently, an involvement of TLRs in various physiologic and pathologic processes has been reported. Because many of these processes implicate angiogenesis, we here elucidated the role of a TLR2/6-dependent pathway on angiogenesis using the TLR2/6 agonist macrophage-activating lipopeptide of 2 kDa (MALP-2), a common bacterial lipopeptide. In vivo and in vitro Matrigel assays demonstrated that MALP-2 promoted angiogenesis in a TLR2/6-dependent manner. Moreover, MALP-2 induced endothelial cell proliferation and migration and a strong secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF release in response to MALP-2 from isolated vascular segments was completely prevented when the endothelium was removed. MALP-2 containing Matrigel implants exhibited vascular structures as well as CD45(+) cells. MALP-2 induced migration of leukocytes and likewise GM-CSF release, particularly from the monocyte population. Inhibition of GM-CSF by siRNA or antibodies suppressed MALP-2-induced angiogenesis in vitro and in vivo. These results clearly identified a TLR2/6-dependent induction of angiogenesis by the bacterial lipopeptide MALP-2, which is mediated by GM-CSF. This might represent a general mechanism to enhance or restore blood flow and recruit immune cells for pathogen defense and tissue regeneration.
Assuntos
Células Endoteliais/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Neovascularização Fisiológica/fisiologia , Receptor 2 Toll-Like/metabolismo , Receptor 6 Toll-Like/metabolismo , Animais , Anticorpos/farmacologia , Aorta/citologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Humanos , Sistema Imunitário/fisiologia , Leucócitos/citologia , Leucócitos/imunologia , Leucócitos/metabolismo , Lipopeptídeos/metabolismo , Lipopeptídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/efeitos dos fármacos , RNA Interferente Pequeno , Regeneração/fisiologia , Receptor 2 Toll-Like/genética , Receptor 6 Toll-Like/genética , Veias Umbilicais/citologiaRESUMO
Chronic skin ulcers, such as leg ulcers, pressure sores and diabetic foot ulcers, are a challenge to physicians and medical personnel and a cause of tremendous discomfort and ensuing loss of quality of life to the patients. Wound healing involves production and action of various growth factors. A novel approach, distinct from the application of single growth factors, is the administration of the macrophage stimulator macrophage-activating lipopeptide-2 (MALP-2). The rationale is based on the finding that macrophages are the main source of several growth factors required for wound healing, which are sequentially released during this process. MALP-2 has previously been shown to be effective in an established animal model with diabetic mice. The purpose of the present phase I study was to establish tolerability of MALP-2 when applied into small cutaneous wounds in human beings. Twelve patients (six females and six males; mean age 66.8 years; range 52-87 years) with different diagnoses were enrolled into the study. An artificial wound was created with a 2-mm diameter skin biopsy punch and a volume of 30 microl MALP-2 (0.125-1 microg) or vehicle control, respectively, was injected intracutaneously into the wound and closed with a water-resistant transparent adhesive. Photos were taken daily from every patient up to 6 days, and skin biopsies were performed after 1 week from six patients. We could show in the present study for the first time that MALP-2 caused a transient erythema and was tolerated without any systemic side effects up to a dose of 1 microg per wound in human beings. In healthy as well as in diabetic patients, MALP-2 induced local inflammation that faded after 48 h. The effectiveness of MALP-2 in the healing of chronic wounds in humans, e.g. in chronic skin ulcers, such as leg ulcers, pressure sores and diabetic foot ulcers, could now be addressed in further studies.
Assuntos
Lipopeptídeos/uso terapêutico , Cicatrização/efeitos dos fármacos , Ferimentos e Lesões/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Quimiocina CCL2/metabolismo , Colágeno Tipo IV/metabolismo , Eritema/induzido quimicamente , Feminino , Humanos , Injeções Intradérmicas , Lipopeptídeos/administração & dosagem , Lipopeptídeos/efeitos adversos , Masculino , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Resultado do Tratamento , Ferimentos e Lesões/metabolismo , Ferimentos e Lesões/patologiaRESUMO
Dendritic cells (DC) modulate immune responses depending on the nature of the antigens. Receptors capable of discriminating these antigens on the basis of the pathogen-associated molecular patterns (PAMP) belong to the Toll-like receptor (TLR) family. The macrophage-activating lipopeptide 2 kDa (MALP-2), a synthetic lipopeptide derived from Mycoplasma fermentans, signals through TLR-2 and TLR-6. The aim of this study was to examine whether MALP-2 can modulate the functional properties of human monocyte-derived DC. The effects of this treatment were compared to those of the TLR-4 agonist lipopolysaccharide (LPS). To ensure clinical applicability, DC were generated under serum-free conditions. MALP-2 and LPS stimulation induced the expression of CD83 and increased the expressions of CD80, CD86, HLA-ABC and CD40. Furthermore, both substances decreased the endocytotic capacity of DC and induced the release of bioactive TNF-alpha and IL-10, whereas LPS additionally increased IL-12 release. Pretreatment with both substances boosted the allostimulatory capacity of DC. In a coculture with autologous lymphocytes, either MALP-2 or LPS pretreated DC induced a marked proliferation of lymphocytes, but only DC prestimulated with MALP-2 activated lymphocytes to produce the cytokines IL-4, IL-5 and IFN-gamma. No polarisation of lymphocytes into T-helper (Th)1 or Th2 was detected. These data indicate that MALP-2 is a potential candidate to modulate DC for clinical applications.
Assuntos
Células Dendríticas/efeitos dos fármacos , Mycoplasma/metabolismo , Oligopeptídeos/farmacologia , Antígenos CD/biossíntese , Antígeno B7-1/biossíntese , Antígeno B7-2 , Antígenos CD40/biossíntese , Divisão Celular , Citocinas/biossíntese , Endocitose , Citometria de Fluxo , Humanos , Imunoglobulinas/biossíntese , Imunofenotipagem , Interferon gama/metabolismo , Interleucina-4/metabolismo , Interleucina-5/metabolismo , Lipopeptídeos , Lipopolissacarídeos/metabolismo , Linfócitos/metabolismo , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Células Th1 , Células Th2 , Fatores de Tempo , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Receptor 6 Toll-Like , Receptores Toll-Like , Antígeno CD83RESUMO
We investigated whether an inflammation-dependent activation of the brain occurs in response to systemic intraperitoneal (i.p.) or local injections of macrophage-activating lipopeptide-2 (MALP-2) into a subcutaneous (s.c.) air pouch, and whether local (peripheral) or central cyclooxygenase (COX)-2-dependent formations of prostaglandin E(2) (PGE(2)) are involved in MALP-2-induced illness responses. Body temperature, activity, food and water intake were measured telemetrically. Local (s.c.) and circulating levels of PGE(2) were measured by an ELISA. Inflammatory activation of the brain in response to MALP-2 was determined by immunohistochemical detection of the transcription factors NFkappaB and STAT3 in cell nuclei as well as the appearance of COX-2 at the same sites. S.c. treatment with the preferential COX-2 inhibitor meloxicam attenuated, but not abolished fever induced by local injections of MALP-2 into the pouch. Local MALP-2-induced formation of PGE(2) was blunted by treatment with meloxicam. In the brain, i.p. stimulation with MALP-2-induced nuclear STAT3- and NFkappaB-translocation in the vasculature and the sensory circumventricular organs, which was accompanied by an increase in COX-2 immunoreactivity (IR) in endothelial cells. Local MALP-2-treatment induced a moderate STAT3 activation and a small but significant increase in COX-2 IR while no NFkappaB-activation could be observed in the brains of these animals. We demonstrated that the activation of the brain STAT3 (NFkappaB)-COX-2 singling cascade seems to be involved in the manifestation of brain-controlled illness symptoms induced by systemic and local inflammatory stimulation with MALP-2. The present data further suggest a contribution of peripherally produced PGE(2) to MALP-2-induced activation of brain sites implicated in fever.
Assuntos
Encéfalo/fisiopatologia , Ciclo-Oxigenase 2/fisiologia , Febre/fisiopatologia , Lipopeptídeos/fisiologia , Animais , Encéfalo/imunologia , Encéfalo/metabolismo , Ciclo-Oxigenase 2/biossíntese , Dinoprostona/biossíntese , Ingestão de Líquidos , Ingestão de Alimentos , Indução Enzimática , Febre/imunologia , Febre/metabolismo , Inflamação/imunologia , Inflamação/metabolismo , Injeções Intraperitoneais , Injeções Subcutâneas , Lipopeptídeos/administração & dosagem , Lipopeptídeos/farmacologia , Masculino , Atividade Motora , NF-kappa B/biossíntese , Ratos , Ratos Wistar , Fator de Transcrição STAT3/biossínteseAssuntos
Síndrome de Behçet/epidemiologia , Síndrome de Behçet/microbiologia , Infecções por Mycoplasma/epidemiologia , Mycoplasma fermentans/imunologia , Adulto , Anticorpos Antibacterianos/sangue , Síndrome de Behçet/imunologia , Feminino , Humanos , Masculino , Infecções por Mycoplasma/imunologia , Estudos SoroepidemiológicosAssuntos
Síndrome de Behçet/complicações , Infecções por Mycoplasma/diagnóstico , Mycoplasma fermentans , Adulto , Anticorpos Antibacterianos/sangue , Síndrome de Behçet/microbiologia , Western Blotting , Feminino , Humanos , Lipopeptídeos , Lipoproteínas/sangue , Lipoproteínas/química , Masculino , Infecções por Mycoplasma/imunologia , Mycoplasma fermentans/isolamento & purificação , Oligopeptídeos/sangue , Oligopeptídeos/química , Valores de ReferênciaRESUMO
Alveolar macrophages (AM) are the primary target cell of the lung for inhaled mycobacterial pathogens. We investigated the effect of the synthetic lipopeptide MALP-2 on the interaction between AM from rats and Mycobacterium bovis BCG. AM were infected with M. bovis BCG at a multiplicity of infection (MOI) of 10 and then cultured in medium alone or medium supplemented with either synthetic macrophage activating lipopeptide-2 (MALP-2), or IFN-gamma, or both. Mycobacterial CFU were counted on days 3 and 7 and cell-free supernatants were collected for cytokine measurements. Treatment of macrophages with MALP-2 led to reduced bacterial loads by day 3 and 7 post-infection and at the same time enhanced the release of TNF-alpha, IL-6 und IL-10 compared to non-stimulated, M. bovis BCG-infected AM. Macrophages co-treated with MALP-2 and IFN-gamma or IFN-gamma in the absence of MALP-2 limited the growth of M. bovis BCG only by day 3, but not day 7 post-stimulation. Our data show that MALP-2 is effective in decreasing bacterial loads in lung sentinel cells.
Assuntos
Lipopeptídeos/farmacologia , Macrófagos Alveolares/microbiologia , Mycobacterium bovis/efeitos dos fármacos , Animais , Contagem de Colônia Microbiana , Citocinas/análise , Interferon gama/farmacologia , Lipopeptídeos/síntese química , Pulmão/microbiologia , Pulmão/patologia , Mycobacterium bovis/crescimento & desenvolvimento , Ratos , Tuberculose/microbiologia , Tuberculose/patologiaRESUMO
Macrophage-activating lipopeptide-2 (MALP-2) from Mycoplasma fermentans has been identified as a pathogen-associated molecular pattern of Mycoplasmas that causes activation of the innate immune system through the activation of the heterodimeric Toll-like receptors (TLRs)-2 and -6. The aim of this study was to characterize the ability of MALP-2 and a synthetic analog fibroblast-stimulating lipopeptide-1 (FSL-1; represents the NH2-terminal sequence of a lipoprotein from M. salivarium) to act as exogenous pyrogens, to induce formation of cytokines (endogenous pyrogens), and to cause sickness behavior, such as depressed motor activity, anorexia, and adipsia. For this purpose, body temperature, activity, food intake, and water intake were recorded for 3 days by use of telemetry devices in several groups of rats treated with MALP-2/FSL-1 or the respective control solutions. Intraperitoneal injections of FSL-1 caused fever at doses of 10 or 100 microg/kg, which was preceded by a pronounced phase of hypothermia in response to a dose of 1,000 microg/kg. The maximal fever (a peak of 1.5 degrees C above baseline) was caused by the 100 microg/kg dose with almost identical responses to both MALP-2 and FSL-1. Fever was accompanied by pronounced rises of the proinflammatory cytokines TNF and IL-6 in plasma. Treatment with the TLR-2 and -6 agonists further induced a dose-dependent manifestation of anorexia and adipsia, as well as a reduction of motor activity. We could thus demonstrate that activation of TLR-2 and -6 can induce systemic inflammation in rats accompanied by the classical signs of brain-controlled illness responses.
Assuntos
Anorexia/induzido quimicamente , Diglicerídeos/farmacologia , Febre/induzido quimicamente , Atividade Motora/efeitos dos fármacos , Oligopeptídeos/farmacologia , Receptor 2 Toll-Like/agonistas , Receptor 6 Toll-Like/agonistas , Animais , Peso Corporal/efeitos dos fármacos , Encéfalo/metabolismo , Ritmo Circadiano , Ingestão de Líquidos/efeitos dos fármacos , Inflamação/induzido quimicamente , Lipopeptídeos , Masculino , Ratos , Ratos WistarRESUMO
RATIONALE: The incidence and prevalence of allergic asthma, caused by Th2-mediated inflammation in response to environmental antigens, is increasing. Epidemiologic data suggest that a lack of Th1-inducing factors may play a pivotal role in the development of this disease. We have previously shown that dendritic cells treated with macrophage-activating lipopeptide-2 (MALP-2) combined with IFN-gamma modulate the Th2 response toward Th1 in an in vitro allergy model. OBJECTIVE: To test in vivo efficacy of this regime, the effects of the substances were evaluated in a mouse model of allergic airway inflammation. METHODS: Female Balb/c mice were sensitized to ovalbumin, whereas control animals were sham-sensitized with adjuvant only. After 4 weeks, MALP-2 and IFN-gamma or NaCl, respectively, were intratracheally instillated. After inhalational ovalbumin challenge, airway hyperreactivity (AHR) to inhaled methacholine was measured by head-out body plethysmography. The animals were subsequently killed to sample bronchoalveolar lavage fluid and lungs. RESULTS: Sensitized NaCl-treated mice developed marked AHR compared with sham-sensitized animals. This coincided with eosinophilia as well as the amplification of eotaxin and the Th2 cytokines interleukin (IL)-5 and IL-13 in the bronchoalveolar lavage fluid. Treatment of sensitized mice with MALP-2 and IFN-gamma significantly reduced AHR compared with the sensitized, NaCl-treated positive control. Eosinophilia as well as Th2 cytokines were reduced to the levels of unsensitized animals. In contrast, IL-12p70 and neutrophils were markedly increased by treatment with both substances. CONCLUSION: These data demonstrate the in vivo efficacy of MALP-2 and IFN-gamma to reduce allergic inflammation and AHR in allergic asthma.
Assuntos
Asma/imunologia , Hiper-Reatividade Brônquica/imunologia , Fatores Imunológicos/administração & dosagem , Interferon gama/administração & dosagem , Oligopeptídeos/administração & dosagem , Administração por Inalação , Animais , Asma/complicações , Asma/tratamento farmacológico , Hiper-Reatividade Brônquica/tratamento farmacológico , Modelos Animais de Doenças , Lipopeptídeos , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Células Th2/imunologiaRESUMO
The innate immune system uses Toll-like receptors (TLRs) to activate and instruct immune responses against microbial pathogens. Administration of TLR agonists to mice induces a state of hyporesponsiveness, or tolerance, characterized by reduced cytokine production upon subsequent second challenge. The present study examined the effects of pre-treatment of mice with TLR2-dependent stimuli on the host defense against acute polymicrobial infection. Immune priming of mice with macrophage-activating lipopeptide-2 (MALP-2) 4 days prior to infection greatly improves survival and bacterial clearance in a model of polymicrobial septic peritonitis which is associated with enhanced accumulation of effector neutrophils in the peritoneal cavity. Further, the systemic and local production of both myeloid differentiation factor 88 (MyD88)-dependently and MyD88-independently produced cytokines was substantially diminished, but not completely absent, in TLR2-treated mice. While pre-treatment with MALP-2 does not involve differential expression of TLR and IL-1R-associated kinase proteins, ST2, a negative regulator of TLR signaling, was up-regulated after treatment of mice with either MALP-2 or N-alpha-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-L-cysteine. Therefore, ST2 may be a mechanism, among others, to attenuate the sepsis-induced cytokine burst. Thus, these results suggest that immune protection in mice after pre-treatment with TLR2-dependent stimuli involves the induction of enhanced pathogen defense by neutrophils. In addition, up-regulation of ST2 could contribute to the diminished cytokine production.
Assuntos
Proteínas de Membrana/imunologia , Peritonite/imunologia , Peritonite/prevenção & controle , Receptores Imunológicos/agonistas , Animais , Sequência de Bases , Quimiocinas/biossíntese , Cisteína/análogos & derivados , Cisteína/farmacologia , Citocinas/biossíntese , DNA Complementar/genética , Feminino , Quinases Associadas a Receptores de Interleucina-1 , Proteína 1 Semelhante a Receptor de Interleucina-1 , Lipopeptídeos , Lipoproteínas/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Oligopeptídeos/farmacologia , Peritonite/etiologia , Proteínas Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Imunológicos/genética , Receptores de Interleucina , Transdução de Sinais , Receptor 2 Toll-Like , Regulação para Cima/efeitos dos fármacosRESUMO
The macrophage-activating lipopeptide-2 (MALP-2) is an agonist of the TLR heterodimer 2/6, which exhibits potent activity as mucosal adjuvant, promoting strong humoral and cellular responses. Although B cells expressing TLR2/6 are potential targets, very little is known about the effect of MALP-2 on B cells. Studies were performed using total spleen cells or purified B cells from WT mice or animals deficient in TLR2, T cells, B cells, or specific subpopulations of B cells. They demonstrated that MALP-2 promotes a T cell-independent activation and maturation of B cells (mainly follicular but also B-1a and marginal zone B cells) via TLR2. MALP-2 also increased the frequency of IgM- and IgG-secreting cells, but bystander cells were required for IgA secretion. Activated B cells exhibited increased expression of activation markers and ligands that are critical for cross-talk with T cells (CD19, CD25, CD80, CD86, MHC I, MHC II, and CD40). Immunization of mice lacking T cells showed that MALP-2-mediated stimulation of TLR2/6 was unable to circumvent the need of T cell help for efficient Ag-specific B cell activation. Immunization of mice lacking B cells demonstrated that B cells are critical for MALP-2-dependent improvement of T cell responses. The knowledge emerging from this work suggests that MALP-2-mediated activation of B cells through TLR2/6 is critical for adjuvanticity. B cell stimulation by pattern recognition receptors seems to be a basic mechanism that can be exploited to improve the immunogenicity of vaccine formulations.
Assuntos
Adjuvantes Imunológicos/fisiologia , Células Apresentadoras de Antígenos , Linfócitos B/imunologia , Lipoproteínas/fisiologia , Ativação Linfocitária/imunologia , Ativação de Macrófagos/imunologia , Oligopeptídeos/fisiologia , Receptores Imunológicos/fisiologia , Animais , Células Apresentadoras de Antígenos/imunologia , Linfócitos B/metabolismo , Biomarcadores/metabolismo , Proliferação de Células , Células Cultivadas , Imunidade nas Mucosas/genética , Lipopeptídeos , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Baço/citologia , Baço/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Receptor 2 Toll-LikeRESUMO
Patients or experimental animals previously exposed to lipopolysaccharide (LPS) become tolerant to further LPS challenge. We investigated the potential of the macrophage-activating lipopeptide 2 (MALP-2) to induce in vivo cross tolerance to tumor necrosis factor alpha (TNF-alpha) and LPS. MALP-2-induced tolerance could be of practical interest, as MALP-2 proved much less pyrogenic in rabbits than LPS. Whereas LPS signals via Toll-like receptor 4 (TLR4), MALP-2 uses TLR2 and TLR6. LPS-mediated cytokine release was studied in mice pretreated with intraperitoneal injections of MALP-2. No biologically active TNF-alpha could be detected in the serum of MALP-2-treated animals when challenged with LPS 24 or 72 h later, whereas suppression of LPS-dependent interleukin (IL)-6 lasted for only 24 h. Protection from lethal TNF-alpha shock was studied in galactosamine-treated mice. Dose dependently, MALP-2 prevented death from lethal TNF-alpha doses in TLR4(-/-) but not in TLR2(-/-) mice, with protection lasting from 5 to 24 h. To assay protection from LPS, mice were pretreated with MALP-2 doses of up to 10 micro g. Five and 24 h later, the animals were simultaneously sensitized and challenged by intravenous coinjection of galactosamine and a lethal dose of 50 ng of LPS. There was only limited protection (four of seven mice survived) when mice were challenged 5 h after MALP-2 pretreatment, and no protection when mice were challenged at later times. The high effectiveness of MALP-2 in suppressing TNF-alpha, the known ways of biological inactivation, and low pyrogenicity make MALP-2 a potential candidate for clinical use.
Assuntos
Lipopolissacarídeos/toxicidade , Glicoproteínas de Membrana/fisiologia , Oligopeptídeos/farmacologia , Receptores de Superfície Celular/fisiologia , Fator de Necrose Tumoral alfa/toxicidade , Animais , Tolerância a Medicamentos , Feminino , Galactosamina/farmacologia , Humanos , Lipopeptídeos , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oligopeptídeos/toxicidade , Pirogênios/toxicidade , Transdução de Sinais , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Receptores Toll-Like , Fator de Necrose Tumoral alfa/biossínteseRESUMO
The question which detailed structures of bacterial modulins determine their relative biological activity and respective host cell receptors was examined with synthetic variants of mycoplasmal lipopeptides as model compounds, as well as recombinant outer surface protein A (OspA) of Borrelia burgdorferi and lipoteichoic acid. Mouse fibroblasts bearing genetic deletions of various toll-like receptors (TLR) were the indicator cells to study receptor requirements, primary macrophages served to measure dose response. The following results were obtained: (i) the TLR system discriminates between modulins with three and those with two long-chain fatty acids in their lipid moiety, in that lipopeptides with three fatty acids were recognized by TLR2, whereas those with two long-chain fatty acids and lipoteichoic acid required the additional cooperation with TLR6; (ii) substitution of the free N terminus of mycoplasmal lipopeptides with an acetyl or palmitoyl group decreased the specific activity; (iii) removal of one or both ester-bound fatty acids lowered the specific activity by five orders of magnitude or deleted biological activity; (iv) oxidation of the thioether group lowered the specific activity by at least four orders of magnitude. The implications of these findings for physiological inactivation of lipopeptides and host-bacteria interactions in general are discussed.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Drosophila , Lipoproteínas/química , Lipoproteínas/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Borrelia/química , Feminino , Fibroblastos/imunologia , Fibroblastos/metabolismo , Lipopeptídeos , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Lipoproteínas/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estrutura Molecular , Oligopeptídeos/química , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Ácidos Teicoicos/química , Ácidos Teicoicos/metabolismo , Receptor 2 Toll-Like , Receptor 6 Toll-Like , Receptores Toll-LikeRESUMO
Dendritic cells (DC) are the most potent APCs with the capacity to induce, modulate, or shut down immune function. These features make them potentially useful for treating diseases associated with misled immunologic responses. Therefore, it was the aim of this study to reverse the allergen-dependent Th2 reaction responsible for allergic symptoms by modulating DC function. This issue was addressed in an in vitro test system consisting of human monocyte-derived allergen-pulsed DC from allergics cocultured with autologous lymphocytes. A Th2 reaction judged by the amplification of IL-4 and the down-regulation of IFN-gamma was induced by pulsing DC with the relevant allergen. To modulate this reaction, the Toll-like receptor 2/6 engaging mycoplasmal lipopetide macrophage-activating lipopeptide 2 kDa was combined with IFN-gamma to stimulate allergen-pulsed DC. Such treatment resulted in a 500-fold increase in IFN-gamma production in the supernatant of cocultured autologous lymphocytes, while the Th2 marker IL-4 was not affected. This phenomenon was associated with an increase in proliferation and the number of IFN-gamma-producing lymphocytes. Phenotype and function of thus treated DC remained stable. These data indicate that a former allergen-dependent Th2 reaction can be reversed toward a Th1-type response by an appropriate treatment of DC.
Assuntos
Proteínas de Bactérias/fisiologia , Hipersensibilidade Imediata/imunologia , Interferon gama/fisiologia , Ativação de Macrófagos , Glicoproteínas de Membrana/agonistas , Oligopeptídeos/fisiologia , Receptores de Superfície Celular/agonistas , Células Th2/imunologia , Antígenos de Dermatophagoides/imunologia , Proteínas de Artrópodes , Divisão Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Cisteína Endopeptidases , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Combinação de Medicamentos , Sinergismo Farmacológico , Humanos , Interferon gama/biossíntese , Interleucina-12/biossíntese , Interleucina-12/fisiologia , Interleucina-4/biossíntese , Lipopeptídeos , Subpopulações de Linfócitos/citologia , Subpopulações de Linfócitos/imunologia , Subpopulações de Linfócitos/metabolismo , Subunidades Proteicas/biossíntese , Subunidades Proteicas/fisiologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/metabolismo , Receptor 2 Toll-Like , Receptor 6 Toll-Like , Receptores Toll-LikeRESUMO
The adjuvanticity of MALP-2, a 2-kDa synthetic lipopeptide with macrophage-stimulatory activity, was evaluated in BALB/c mice using beta-galactosidase (beta-gal) as model antigen. When co-administered with beta-gal by either the intranasal (i.n.) or i.p. route, MALP-2 (0.5 microg) was capable of increasing beta-gal-specific serum IgG titers by 675-3,560-fold (i.n.) and 64-128-fold (i.p.), respectively, as compared to immunization with beta-gal alone. Using MALP-2, almost maximal IgG responses were already stimulated following the first immunization, and the IgG titers were similar to those observed using 10 microg of cholera toxin B subunit (CTB) as adjuvant. The mucosal immune system was also effectively stimulated (p<0.05) when MALP-2 was administered by the i.n. route (36% and 23% of beta-gal-specific IgA in lung and vaginal lavages, respectively). The i.n. co-administration of MALP-2 stimulated a stronger cellular immune response than CTB, both in submandibular lymph nodes and spleen (p<0.05). The analysis of beta-gal-specific IgG isotypes and the profiles of cytokines secreted by in vitro re-stimulated cells showed that co-administration of MALP-2 triggered a dominant Th2-response pattern. A recruitment of B220(+) and MAC-1(+) cells with an up-regulated expression of MHC class I, CD80 (B7.1) and CD54 (ICAM-1) was observed in nasal associated lymphoid tissues from MALP-2 treated mice. Taken together, our results demonstrated that the synthetic lipopeptide MALP-2 represents a very promising adjuvant for the mucosal delivery of vaccine antigens.
Assuntos
Adjuvantes Imunológicos/farmacologia , Proteínas de Bactérias/farmacologia , Lipoproteínas/farmacologia , Mycoplasma , Oligopeptídeos/farmacologia , Administração Intranasal , Animais , Formação de Anticorpos/efeitos dos fármacos , Proteínas de Bactérias/imunologia , Feminino , Imunidade nas Mucosas , Lipopeptídeos , Lipoproteínas/imunologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Oligopeptídeos/imunologiaRESUMO
Mycoplasmas can cause interstitial pneumonias inducing critical illness in humans and animals. Mycoplasma infections are characterized by an influx of neutrophils, followed by an accumulation of macrophages and lymphocytes. The present study deals with the question of which mycoplasmal components cause this host reaction. The mycoplasma-derived, macrophage-activating lipopeptide 2S-MALP-2 was used to mimic the sequelae of a mycoplasma infection. To this end, 2S-MALP-2 was intratracheally instilled into the lungs of Lewis rats, and the bronchoalveolar lavage cells were examined at different times after different doses of 2S-MALP-2. Application of 2.5 microg induced a pronounced leukocyte accumulation in the bronchoalveolar space. At 24 h after 2S-MALP-2 administration, the majority of leukocytes consisted of neutrophils, followed by macrophages, peaking on days 2 and 3. Lymphocyte numbers, although amounting to only a few percent of the total bronchoalveolar lavage cells, also increased significantly, with maximal lymphocyte accumulation occurring by 72 h after instillation. The leukocyte count of the lung interstitium was increased on day 3 after treatment. After 10 days all investigated cell populations returned to control levels. Transient chemotactic activity for neutrophils was detected in the bronchoalveolar lavage fluid early after 2S-MALP-2 application, followed by monocyte chemoattractant protein-1 activity (MCP-1) in lung homogenates. MCP-1 was produced by bronchoalveolar lavage cells upon stimulation with 2S-MALP-2. Our data indicate that mycoplasmal lipoproteins and lipopeptides are probably the most relevant mycoplasmal components for the early host reaction. The primary target cells are likely to be the alveolar macrophages liberating chemokines, which attract further leukocytes.
Assuntos
Proteínas de Bactérias/imunologia , Lipoproteínas/imunologia , Pulmão/imunologia , Ativação de Macrófagos/imunologia , Macrófagos Alveolares/imunologia , Mycoplasma fermentans/imunologia , Oligopeptídeos/imunologia , Animais , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/síntese química , Lavagem Broncoalveolar , Contagem de Células , Quimiocina CCL2/imunologia , Quimiocina CCL5/administração & dosagem , Quimiotaxia de Leucócito/imunologia , Relação Dose-Resposta a Droga , Interleucina-16/administração & dosagem , Leucócitos/citologia , Leucócitos/imunologia , Lipopeptídeos , Lipoproteínas/administração & dosagem , Lipoproteínas/síntese química , Linfócitos/citologia , Macrófagos Alveolares/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Oligopeptídeos/administração & dosagem , Oligopeptídeos/síntese química , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Estereoisomerismo , Fatores de Tempo , Traqueia/imunologiaRESUMO
A major requirement for HIV/AIDS research is the development of a mucosal vaccine that stimulates humoral and cell-mediated immune responses at systemic and mucosal levels, thereby blocking virus replication at the entry port. Thus, a vaccine prototype based on biologically active HIV-1 Tat protein as antigen and the synthetic lipopeptide, macrophage-activating lipopeptide-2 (MALP-2), asa mucosal adjuvant was developed. Intranasal administration to mice stimulated systemic and mucosal anti-Tat antibody responses, and Tat-specific T cell responses, that were more efficient than those observed after i.p. immunization with Tat plus incomplete Freund's adjuvant. Major linear B cell epitopes mapped within aa 1-20 and 46-60, whereas T cell epitopes were identified within aa 36-50 and 56-70. These epitopes have also been described in vaccinated primates and in HIV-1-infected individuals with better prognosis. Analysis of the anti-Tat IgG isotypes in serum, and the cytokine profile of spleen cells indicated that a dominant Th1 helper response was stimulated by Tat plus MALP-2, as opposed to the Th2 response observed with Tat plus incomplete Freund's adjuvant. Tat-specific IFN-gamma-producing cells were significantly increased only in response to Tat plus MALP-2. These data suggest that Malp-2 may represent an optimal mucosal adjuvant for candidate HIV vaccines based on Tat alone or in combination with other HIV antigens.