C型凝集素受体对固有免疫应答的双向调节作用

C型凝集素受体（CLR）是模式识别受体家族中有别于TLR的新家族,在机体免疫应答中发挥着重要作用。近年来,人们对CLR对固有免疫应答的调节作用进行了较为广泛的研究,越来越多的研究发现CLRs对固有免疫应答具有双向调节作用：不同的CLR在固有免疫应答中既可以发挥正性调节作用,也能够发挥负性调节作用;在针对不同的病原体或在抗原提呈细胞不同的成熟状态等条件下,同-CLR在固有免疫应答中也可以发挥正负调节两方面的作用。深入探讨CLR对固有免疫应答的双向调节作用,对于进一步理解免疫应答的精细调节及疾病发病机制的复杂性等都有重要的理论意义,同时也为许多疾病的预防与治疗提供了一种新的策略。     

Protein/peptide and DNA vaccine delivery by targeting C-type lectin receptors

C-type lectin receptors (CLRs) are a class of pathogen-recognition receptors that are actively investigated in the field of vaccine delivery. Many of their properties have functions linked to the immune system. These receptors are expressed abundantly on antigen-presenting cells and are considered to be the sentinels of immune surveillance owing to their endocytic nature and the ability to recognize a diverse range of pathogens through recognition of pathogen-associated molecular patterns. CLRs are also involved in the processes of antigen presentation mediated through the induction of dendritic cell maturation and cytokine production. These properties engender CLRs to be ideal for vaccine targeting. Conversely, CLRs also function to recognize glycosylated self-antigens to induce homeostatic control and tolerance. In this review, we will describe the various preclinical/clinical vaccination strategies to target antigens and plasmid DNA to this diverse class of receptors.     

Endogenous ligands for C-type lectin receptors: the true regulators of immune homeostasis

Summary:  C-type lectin receptors (CLRs) have long been known as pattern-recognition receptors implicated in the recognition of pathogens by the innate immune system. However, evidence is accumulating that many CLRs are also able to recognize endogenous 'self' ligands and that this recognition event often plays an important role in immune homeostasis. In the present review, we focus on the human and mouse CLRs for which endogenous ligands have been described. Special attention is given to the signaling events initiated upon recognition of the self ligand and the regulation of glycosylation as a switch modulating CLR recognition, and therefore, immune homeostasis.     

Interleukin-22 produced by alveolar macrophages during activation of the innate immune response

Objective and design

Interleukin (IL)-22 is important for mucosal host defense. Whereas previous studies focus on lymphocytes as sources of IL-22, we determined whether IL-22 is produced by inflammatory cells in the lungs other than T-lymphocytes during the activation of the innate immune response.

Material, methods and treatment

Inflammatory cells in the lungs of Balb/c mice were primed by endotoxin (LPS, 10 μg) or peptidoglycan (PG, 40 μg) intranasally (3 days). After CD3 + cell depletion, lung homogenates were re-stimulated 24 h with LPS (100 ng/ml), PG (10 μg/ml), IL-23 (100 ng/ml) or vehicle. Human BAL macrophages were stimulated 24 h with PG (50 μg/ml) and IL-23 (100 ng/ml) or vehicle. The release of IL-22 was measured with ELISA and intracellular IL-22 with immunostaining. For statistics, either Dunnett or Students t test method was employed (n = 3–8).

Results

Re-stimulation in vitro increased concentrations of mouse IL-22 protein irrespective of priming in vivo. A majority of macrophages in mouse lung and BAL samples displayed immunostaining for IL-22. In analogy, human BAL macrophages released IL-22 protein, and a third of these cells displayed immunostaining for IL-22.

Conclusions

Alveolar macrophages can produce and release IL-22 during the activation of the innate immune response and thereby constitute a potentially important regulator of mucosal host defence in the lungs.     

Modulation of allergen-specific immune responses to the major shrimp allergen, tropomyosin, by specific targeting to scavenger receptors on macrophages

BACKGROUND: Tropomyosin from shrimp is the major cross-reacting crustacean food allergen. Earlier studies have led to the purification and immunochemical characterization of the major IgE binding epitopes of the allergen. Maleylated proteins are known to be specifically targeted to scavenger receptors on macrophage. Since antigens processed and presented by macrophages are known to elicit Th1 type of responses and allergic responses are characterized by polarization towards Th2 phenotype, the possibility of modulation of allergen-specific immune responses by targeting of tropomyosin to macrophage via scavenger receptor was explored. METHODS: The IgG and IgE binding potential of the native maleylated form of tropomyosin was carried out by ELISA and immunoblot. The ability of the native and maleylated form of allergen to induce in vitro proliferation of splenocytes from BALB/C mice immunized with both forms of allergen was tested. The in vitro production of IL-4 and IFN-gamma by splenocytes from mice immunized with the two forms of allergen was determined from culture supernatants. The in vivo production of serum IgG1 and IgG2a antibodies following immunization with native and modified allergens was monitored by ELISA. RESULTS: The maleylated form of tropomyosin was found to have reduced antigenicity and allergenicity as compared to its native counterpart. The modified allergen was, however, found to elicit a cellular response similar to native tropomyosin in vitro. Analysis of the cytokine profiles showed a modulation from an IL-4-dominant, proallergic, Th2 phenotype to an IFN-gamma-dominant, antiallergic, Th1 phenotype that could also be correlated to a modulation in the in vivo antibody isotype. CONCLUSION: The results suggest the possible potential for modulating allergic responses in vivo by selective targeting to macrophages.     

C-type lectins on macrophages participate in the immunomodulatory response to Fasciola hepatica products

Fasciola hepatica releases excretory-secretory products (FhESP), and immunomodulatory properties have been described for the carbohydrates present in these parasite products. The interaction of FhESP with the innate immune cells, such as macrophages, is crucial in the early stage of infection. In this work we observed that peritoneal macrophages from naive BALB/c mice stimulated in vitro with FhESP presented: an increased arginase activity as well as Arginase I expression, and high levels of transforming growth factor-β and interleukin-10. A similar macrophage population was also observed in the peritoneum of infected mice. A partial inhibition of the immunomodulatory effects described above was observed when macrophages were pre-incubated with Mannan, anti-mannose receptor, Laminarin or anti-Dectin-1, and then stimulated with FhESP. In addition, we observed a partial inhibition of these effects in macrophages obtained from mice that were intraperitoneally injected with Mannan or Laminarin before being infected. Taken together, these results suggest the participation of at least two C-type lectin receptors, mannose receptor and Dectin-1, in the interaction of FhESP with macrophages, which allows this parasite to induce immunoregulatory effects on these important innate immune cells and may constitute a crucial event for extending its survival in the host.     

Regulation of immune response to inhaled antigen by alveolar macrophages: differential effects of in vivo alveolar macrophage elimination on the induction of tolerance vs. immunity

A single intratracheal instillation of liposomes containing dichloro methylene diphosphonate into rats eliminated greater than 80% of the alveolar macrophage (AM phi) population, and the population was not significantly renewed during the ensuing week. AM phi depletion markedly increased local antibody production in the lung wall in pre-primed animals exposed to antigen aerosols. However, AM phi depletion did not affect the normal development of protective tolerance (particularly in the IgE antibody class) to inhaled antigen in immunologically naive rats. These results are discussed in relation to regional control of immune responses in the upper vs. the lower respiratory tract.     

SIGN-R1, a novel C-type lectin expressed by marginal zone macrophages in spleen,mediates uptake of the polysaccharide dextran

The marginal zone macrophages of the spleen are implicated in the clearance of polysaccharides, but underlying mechanisms need to be pinpointed. SIGN-R1 is one of five recently identified mouse genes that are homologous to human DC-SIGN and encode a single, external, C-terminal C-type lectin domain. We find that a polyclonal antibody to a specific SIGN-R1 peptide reacts primarily and strongly with a subset of macrophages in the marginal zone of spleen and lymph node medulla. In both sites, SIGN-R1 exists primarily in an aggregated form, resistant to dissociation into monomers upon boiling in SDS under reducing conditions. Upon transfection into three different cell lines, high-mol.-wt forms bearing SIGN-R1 are expressed, as well as reactivity with ER-TR9, a mAb previously described to react selectively with marginal zone macrophages. SIGN-R1-expressing macrophages preferentially sequester dextrans following i.v. injection. Likewise, when phagocytic cells are enriched from spleen and tested in culture, dextran is selectively endocytosed by a subset of very large SIGN-R1(+) cells representing approximately 5% of total released macrophages. Uptake of FITC-dextran by these macrophages in vivo and in vitro is blocked by ER-TR9 and polyclonal anti-SIGN-R1 antibodies. Following transfection with SIGN-R1, cell lines become competent to endocytose dextrans. The dextran localizes primarily to compartments lacking transferrin receptor and the LAMP-1 CD107a panlysosomal antigen. Therefore, SIGN-R1 mediates the uptake of dextran polysaccharides, and it is predominantly expressed in the macrophages of the splenic marginal zone and lymph node medulla.     

Mannan-binding lectin deficiency modulates the humoral immune response dependent on the genetic environment

Mannan-binding lectin (MBL) is a plasma protein implicated in innate immune defence against a broad range of microorganisms, including viruses. It is also thought that MBL plays a role in the recruitment of the specific clonal immune response. This was studied by injecting soluble hepatitis B surface antigen (HBsAg) intravenously into mice deficient in both MBL-A and MBL-C (MBL DKO mice). The MBL DKO animals on mixed genetic background (SV129EvSv × C57BL/6) produced higher antibody titres than the wild-type littermates. After primary challenge with the antigen the immunoglobulin M anti-HBsAg antibody titres were threefold higher in the MBL DKO mice than in the wild-type mice. Following the boost, the immunoglobulin G anti-HBsAg antibody titres were 10-fold higher in the MBL DKO mice, suggesting that MBL plays a role in a negative feedback regulation of adaptive immunity. However, the modulating effect of MBL was dependent on the genetic environment. The MBL DKO mice backcrossed on a C57BL/6 background showed the opposite response with the MBL DKO mice now producing fewer antibodies than the wild-type animals, whereas MBL deficiency in mice with the SV129EvSv background did not show any effect in antibody production. These findings indicate that the modifying effect of MBL on the humoral immune response is influenced by the genetic environment.     

Restoration of the immune response by macrophages in mice after administration of immunodepressants

The possibility of restoring the primary immune response in mice immunized with bacteriophage T2 after administration of various immunodepressants (actinomycin D, olivomycin, rifampicin, chloramphenicol, chloroquine, histone F2a, and bacterial lipopolysaccharide) was studied. For this purpose peritoneal macrophages from intact syngeneic donors were transferred simultaneously with injection of the antigen into the recipients following administration of the immunodepressants. The immunodepressive effects of most of the substances studied could be completely compensated in this way. Macrophages can thus be regarded as one of the sites of action of the immunodepressants studied. The results also indicate a role of the macrophages in the induction of the primary immune response to bacteriophage T2 (synthesis of phage-neutralizing antibodies).     

Regulation of the immune response and inflammation by histamine and histamine receptors

Histamine is a biogenic amine with extensive effects on many cell types, including important immunologic cells, such as antigen-presenting cells, natural killer cells, epithelial cells, and T and B lymphocytes. Histamine and its 4 receptors represent a complex system of immunoregulation with distinct effects dependent on receptor subtypes and their differential expression. These are influenced by the stage of cell differentiation, as well as microenvironmental influences, leading to the selective recruitment of effector cells into tissue sites accompanied by effects on cellular maturation, activation, polarization, and effector functions, which lead to tolerogenic or proinflammatory responses. In this review we discuss the regulation of histamine secretion, receptor expression, and differential activation of cells within both the innate and adaptive immune responses. It is clear that the effects of histamine on immune homeostasis are dependent on the expression and activity of the 4 currently known histamine receptors, and we also recognize that 100 years after the original identification of this biogenic amine, we still do not fully understand the complex regulatory interactions between histamine and the host immune response to everyday microbial and environmental challenges.     

Evasion of innate immune responses: evidence for mannose binding lectin inhibition of tumor necrosis factor alpha production by macrophages in response to Blastomyces dermatitidis

Serum factors, including mannose binding lectins (MBL), influence innate responses to microbes. Little is known about the effects of serum factors or MBL on the interaction of Blastomyces dermatitidis, a pulmonary fungal pathogen, with macrophages or on tumor necrosis factor alpha (TNF-α) production. Since macrophage production of TNF-α is an important innate immune response, we examined a mouse peritoneal macrophage (PM) cell line (RAW) and resident PM from CD-1 mice to study TNF-α production by PM stimulated with heat-killed (HK) or live B. dermatitidis yeast cells. Mouse serum and heat-inactivated mouse serum inhibited TNF-α production 94% when macrophages were stimulated by B. dermatitidis, whereas mouse immunoglobulin G (IgG) did not have this effect. HK B. dermatitidis incubated with serum and then washed also failed to stimulate significant TNF-α production by PM. By the sandwich immunofluorescent antibody (IFA) method with anti-mouse MBL (MBL-A or -C), we showed that serum MBL bound to B. dermatitidis. When serum was absorbed with HK B. dermatitidis or live B. dermatitidis, absorbed serum failed to significantly inhibit TNF-α production by RAW cells plus B. dermatitidis, and immunoblotting showed that absorbed serum was depleted of MBL-C. If serum was absorbed with live B. dermatitidis, unbound serum was eluted, and bound serum factor(s) (BS) was released with guanidine buffer, BS inhibited TNF-α production by PM plus B. dermatitidis in a concentration-dependent manner. BS contained MBL-C, which bound B. dermatitidis, as shown by IFA assay. 1,3-β-Glucan stimulated TNF-α production by PM, and this was inhibited by mouse serum. Treatment of B. dermatitidis with anti-1,3-β-glucan antibody inhibited TNF-α production by PM. With anti-1,3-β-glucan antibody, we showed by IFA assay that B. dermatitidis contained 1,3-β-glucan. In an IFA study with B. dermatitidis, serum with an anti-mouse IgG conjugate did not result in fluorescence, yet serum blocked IFA staining of B. dermatitidis by anti-1,3-β-glucan IgG antibody. This indicated that non-IgG serum factors binding to B. dermatitidis prevented access to 1,3-β-glucan by anti-1,3-β-glucan antibody. These results suggest that the mechanism of inhibition of the innate proinflammatory immune response of PM to B. dermatitidis is mediated by serum MBL binding to B. dermatitidis at 1,3-β-glucan sites or sterically masking 1,3-β-glucan sites, thus preventing 1,3-β-glucan stimulation of PM for TNF-α production.     

Effect of cytofectins on the immune response of murine macrophages to mammalian DNA

DNA, depending on base sequence, can induce a wide range of immune responses. While bacterial DNA is stimulatory, mammalian DNA is inactive alone and can, moreover, inhibit the response to bacterial DNA. To determine whether the mode of cell entry affects the immune properties of mammalian DNA, we have investigated the effects of the cytofectin agents Fugene 6 (Roche Diagnostics Corp., Indianapolis, IN), Lipofectin and Lipofectamine (Life Technologies, Grand Island, NY) on the responses of murine macrophages to DNA from calf thymus and human placenta. Whereas calf thymus and human placenta DNA alone failed to stimulate J774 or RAW264.7 cell lines or bone marrow-derived macrophages, these DNAs in complexes with cytofectin agents stimulated macrophages to produce nitric oxide but not interleukin 12. Both single-stranded and double-stranded DNAs were active in the presence of cytofectins. Macrophage activation by the DNA-cytofectin complexes was reduced by chloroquine, suggesting a role of endosomal acidification in activation. As shown by flow cytometry and confocal microscopy, the cytofectins caused an increase in the uptake of DNA into cells. Our findings indicate that macrophages vary in their response to DNA depending on uptake pathway, suggesting that activation by DNA reflects not only sequence but also context or intracellular location.     

Effect of various antisera on intensity of the immune response during induction of antibody formation by allogeneic macrophages

Data on the effect of various antisera on the induction of antibody formation by immune allogeneic macrophages are described. A considerable decrease in the intensity of the immune response was observed after injection both of allogeneic antiserum and of antimacrophagal serum during the first 2 days after transplantation of the allogeneic macrophages. Injection of these sera on the following days had no significant effect on the intensity of the immune response. Antierythrocytic serum prevented the accumulation of antibody-forming cells if injected at various times after transplantation of the allogeneic macrophages.Institute of Medical Genetics, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR P. D. Gorizontov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 9, pp. 1094–1096, September, 1976.     

The promotion of mandibular defect healing by the targeting of S1P receptors and the recruitment of alternatively activated macrophages

Endogenous signals originating at the site of injury are involved in the paracrine recruitment, proliferation, and differentiation of circulating progenitor and diverse inflammatory cell types. Here, we investigate a strategy to exploit endogenous cell recruitment mechanisms to regenerate injured bone by local targeting and activation of sphingosine-1-phosphate (S1P) receptors. A mandibular defect model was selected for evaluating regeneration of bone following trauma or congenital disease. The particular challenges of mandibular reconstruction are inherent in the complex anatomy and function of the bone given that the area is highly vascularized and in close proximity to muscle. Nanofibers composed of poly(dl-lactide-co-glycolide) (PLAGA) and polycaprolactone (PCL) were used to delivery FTY720, a targeted agonist of S1P receptors 1 and 3. In vitro culture of bone progenitor cells on drug-loaded constructs significantly enhanced SDF1α mediated chemotaxis of bone marrow mononuclear cells. In vivo results show that local delivery of FTY720 from composite nanofibers enhanced blood vessel ingrowth and increased recruitment of M2 alternatively activated macrophages, leading to significant osseous tissue ingrowth into critical sized defects after 12 weeks of treatment. These results demonstrate that local activation of S1P receptors is a regenerative cue resulting in recruitment of wound healing or anti-inflammatory macrophages and bone healing. Use of such small molecule therapy can provide an alternative to biological factors for the clinical treatment of critical size craniofacial defects.     

Adjuvant effects of mannose-binding lectin ligands on the immune response to infectious bronchitis vaccine in chickens with high or low serum mannose-binding lectin concentrations

Mannose-binding lectin (MBL) plays a major role in the immune response as a soluble pattern-recognition receptor. MBL deficiency and susceptibility to different types of infections have been subject to extensive studies over the last decades. In humans and chickens, several studies have shown that MBL participates in the protection of hosts against virus infections. Infectious bronchitis (IB) is a highly contagious disease of economic importance in the poultry industry caused by the coronavirus infectious bronchitis virus (IBV). MBL has earlier been described to play a potential role in the pathogenesis of IBV infection and the production of IBV-specific antibodies, which may be exploited in optimising IBV vaccine strategies. The present study shows that MBL has the capability to bind to IBV in vitro. Chickens from two inbred lines (L10H and L10L) selected for high or low MBL serum concentrations, respectively, were vaccinated against IBV with or without the addition of the MBL ligands mannan, chitosan and fructooligosaccharide (FOS). The addition of MBL ligands to the IBV vaccine, especially FOS, enhanced the production of IBV-specific IgG antibody production in L10H chickens, but not L10L chickens after the second vaccination. The addition of FOS to the vaccine also increased the number of circulating CD4+ cells in L10H chickens compared to L10L chickens. The L10H chickens as well as the L10L chickens also showed an increased number of CD4−CD8α−γδ T-cells when an MBL ligand was added to the vaccine, most pronouncedly after the first vaccination. As MBL ligands co-administered with IBV vaccine induced differences between the two chicken lines, these results indirectly suggest that MBL is involved in the immune response to IBV vaccination. Furthermore, the higher antibody response in L10H chickens receiving vaccine and FOS makes FOS a potential adjuvant candidate in an IBV vaccine.