HMGN1 is a novel alarmin that signals through TLR4 and is required for LPS-induced immune responses in vivo.

Neutrophils are the first major population of leukocyte to infiltrate infected or injured tissues and are crucial for initiating host innate defense and adaptive immunity. Although the contribution of neutrophils to innate immune defense is mediated predominantly by phagocytosis and killing of microorganisms, neutrophils also participate in the induction of adaptive immune responses. At sites of infection and/or injury, neutrophils release numerous mediators upon degranulation or death, among these are alarmins which have a characteristic dual capacity to mobilize and activate antigen-presenting cells. We describe here how alarmins released by neutrophil degranulation and/or death can link neutrophils to dendritic cells by promoting their recruitment and activation, resulting in the augmentation of innate and adaptive immune responses.

Granulysin (GNLY), IntroductionThe granulysin (GNLY) gene is located on chromosome 2 and consists of 6 exons. A major protein product of 15 kDa is translated, some of which is subsequently secreted or processed by proteolytic cleavage at both N and C termini to a 9-kDa protein stored in the granular compartment. 1 Whereas the 15-kDa protein is produced rapidly, has a shorter half-life, and is constitutively released, the secretory 9-kDa form is produced slowly and is relatively stable. The crystal structure of the human protein, elucidated in 2003 by Anderson et al, is predictive of its antimicrobial effects and clinical relevance in disease and pathogenesis. GNLY is highly cationic and folded as a 5-helix bundle stabilized by 2 highly conserved intramolecular disulfide bonds. 2 It belongs to the family of Saposin-like lipid binding proteins called SAPLIP and colocalizes in the granular compartments of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells along with granzymes and perforin. 3 GNLY has the highest sequence identity to NK-lysin (43% identity), a porcine protein with antibacterial activity that is also a member of the SAPLIP family. 3 No homologous protein has been identified to date in mice.GNLY is produced by human NK cells and activated CTLs, and the 9-kDa form is rapidly released upon degranulation. The kinetics of expression of GNLY by these 2 cell types differs. NK cells release GNLY very early in immune responses, whereas CTLs release it after 3-5 days of activation. 1 Elevation of GNLY expression and levels in tissue and serum has been reported in infections, autoimmune diseases, transplant rejection, and graft versus host reaction in patients with hematopoietic stem cell transplantation. [4][5][6][7][8][9][10] In leprosy, CD4 ϩ cells expressing GNLY were elevated (8%-15%) in the skin lesions of patients with the tuberculoid form compared with those with the disseminated lepromatous form of the disease. 7 Recent studies have demonstrated that in response to Mycobacterium tuberculosis-infected macrophages, human CD8 ϩ T cells were induced to express CCL5 and GNLY. 11 Recently, it was reported that blister fluids of patients suffering from Stevens Johnson syndrome and toxic epidermal necrolysis contained high levels of secretory 15-kDa GNLY, and the high concentration of GNLY, but not granzyme B or perforin, is responsible for the disseminated keratinocyte apoptosis in Stevens Johnson syndrome and toxic epidermal necrolysis. 12 By contrast, patients with severe immunodeficiencies have very low GNLY serum levels. 13 GNLY levels are also reduced in different cell types and even in serum of carcinoma patients and appear to be inversely correlated with tumor progression. 14-17 Consequently both forms of GNLY can be induced in the course of inflammation and have also been shown to be up-regulated by stimulation of lymphoid cells by proinflammatory mediators and pathogenassociated molecular patterns. 18,19 GNLY exhibits lytic activity against a variety of microorganisms and tumors. 3,14,[20][...

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Lactoferrin is an 80-kDa iron-binding protein present at high concentrations in milk and in the granules of neutrophils. It possesses multiple activities, including antibacterial, antiviral, antifungal, and even antitumor effects. Most of its antimicrobial effects are due to direct interaction with pathogens, but a few reports show that it has direct interactions with cells of the immune system. In this study, we show the ability of recombinant human lactoferrin (talactoferrin alfa (TLF)) to chemoattract monocytes. What is more, addition of TLF to human peripheral blood or monocyte-derived dendritic cell cultures resulted in cell maturation, as evidenced by up-regulated expression of CD80, CD83, and CD86, production of proinflammatory cytokines, and increased capacity to stimulate the proliferation of allogeneic lymphocytes. When injected into the mouse peritoneal cavity, lactoferrin also caused a marked recruitment of neutrophils and macrophages. Immunization of mice with OVA in the presence of TLF promoted Th1-polarized Ag-specific immune responses. These results suggest that lactoferrin contributes to the activation of both the innate and adaptive immune responses by promoting the recruitment of leukocytes and activation of dendritic cells.

Defensins are endogenous, small, cysteine-rich antimicrobial peptides that are produced by leukocytes and epithelial cells. Substantial evidence accumulated in recent years indicates that mammalian defensins are multifunctional and, by interacting with host cell receptor(s), participate in both the innate and adaptive antimicrobial immunity of the host. A better understanding of the function of defensins in immunity has implications for the development of potential clinical therapeutics for the treatment of infection or cancer. Here we will briefly outline the classification, genes, expression, and structure of mammalian defensins and focus on their roles in innate and adaptive immune response of the host.

Alarmins are a group of structurally diverse host defense antimicrobial peptides that are important immune activators. Here we present a novel role of two potent alarmins, human beta defensin 2 and 3 (HBD2 and 3) in promoting IFN-α production by human plasmacytoid DCs (pDCs). We demonstrate that HBD2 and 3 activate pDCs by enhancing the intracellular uptake of CpG and self DNA and promote DNA induced IFN-α production in a TLR9 dependent manner. Both CpG and host DNA form aggregates that resemble DNA nets when combined with HBD2 and 3. Isothermal Titration Calorimetry (ITC) studies to elucidate the nature of HBD3-CpG complexes demonstrates involvement of enthalpy driven interactions in addition to hydrophobic interactions with the formation of complexes at a molar ratio of 2:1 defensin/CpG. Intravenous administration of HBD3-CpG complexes induced proinflammatory cytokines like IL-12, IFN-γ, IL-6, IFN-α and IL-10 in serum associated with an increased recruitment of antigen presenting cells (APCs) in the spleen. Subcutaneous injections of these complexes showed enhanced infiltration of inflammatory cells at injection site indicating a potential pathophysiological role of alarmin/DNA complexes in contributing to inflammation. Intraperitoneal immunization of HBD3/CpG complexes with OVA enhanced both cellular and humoral responses in response to OVA as compared to OVA/HBD3 or OVA/CPG alone, indicative of a much more potent adjuvant effect of the HBD3/CpG complexes. Thus the ability of defensins to enhance cellular uptake of nucleic acids can lead to improved vaccine formulations by promoting their uptake by various cells resulting in an enhanced immune response.

Dendritic cells play a pivotal role in host immune defense, such as elimination of foreign pathogen and inhibition of tumorigenesis. In this paper, we report that [Gd@C 82 (OH) 22 ] n could induce phenotypic maturation of dendritic cells by stimulating DC production of cytokines including IL-12p70, upregulating DC costimulatory (CD80, CD83, and CD86) and MHC (HLA-A,B,C and HLA-DR) molecules, and switching DCs from a CCL5-responsive to a CCL19-responsive phenotype. We found that [Gd@C 82 (OH) 22 ] n can induce dendritic cells to become functionally mature as illustrated by their capacity to activate allogeneic T cells. Mice immunized with ovalbumin in the presence of [Gd@C 82 (OH) 22 ] n exhibit enhanced ovalbumin-specific Th1-polarized immune response as evidenced by the predominantly increased production of IFNγ, IL-1β, and IL-2. The [Gd@C 82 (OH) 22 ] n nanoparticle is a potent activator of dendritic cells and Th1 immune responses. These new findings also provide a rational understanding of the potent anticancer activities of [Gd@C 82 (OH) 22 ] n nanoparticles reported previously.