Atsuko Itakura

Cutaneous Immunization Rapidly Activates Liver Invariant Vα14 NKT Cells Stimulating B-1 B Cells to Initiate T Cell Recruitment for Elicitation of Contact Sensitivity

T cell recruitment to elicit contact sensitivity (CS) requires a CS-initiating process mediated by B-1 cells that produce IgM, which activates complement to promote T cell passage into the tissues. We now show that Vα14i NKT cells induce B-1 cell activation likely by releasing IL-4 early postimmunization. The CS initiation process is absent in Jα18−/− and CD1d−/− NKT cell–deficient mice and is reconstituted by populations enriched for Vα14i NKT cells. Transfers are not effective if cells are derived from IL-4−/− mice. Staining with specific tetramers directly showed that hepatic Vα14i NKT cells increase by 30 min and nearly double by 2 h postimmunization. Transfer of immune B-1 cells also reconstitutes CS responses in NKT cell–deficient mice. The B-1 cells act downstream of the Vα14i NKT cells to restore CS initiation. In addition, IL-4 given systemically to Jα18−/− or CD1d−/− NKT cell–deficient mice reconstitutes elicitation of CS. Further, splenocytes from immune Jα18−/− mice produce less antigen (Ag)-specific IgM antibodies compared with sensitized WT mice. Together these findings indicate that very early after skin immunization Vα14i NKT cells are stimulated to produce IL-4, which activates B-1 cells to produce Ag-specific IgM, subsequently needed to recruit effector T cells for elicitation of CS responses.

B cell-dependent T cell responses: IgM antibodies are required to elicit contact sensitivity.

Contact sensitivity (CS) is a classic example of in vivo T cell immunity in which skin sensitization with reactive hapten leads to immunized T cells, which are then recruited locally to mediate antigen-specific inflammation after subsequent skin challenge. We have previously shown that T cell recruitment in CS is triggered by local activation of complement, which generates C5a that triggers C5a receptors most likely on mast cells. Here, we show that B-1 cell–derived antihapten IgM antibodies generated within 1 day (d) of immunization combine with local challenge antigen to activate complement to recruit the T cells. These findings overturn three widely accepted immune response paradigms by showing that (a) specific IgM antibodies are required to initiate CS, which is a classical model of T cell immunity thought exclusively due to T cells, (b) CS priming induces production of specific IgM antibodies within 1 d, although primary antibody responses typically begin by day 4, and (c) B-1 cells produce the 1-d IgM response to CS priming, although these cells generally are thought to be nonresponsive to antigenic stimulation. Coupled with previous evidence, our findings indicate that the elicitation of CS is initiated by rapidly formed IgM antibodies. The IgM and challenge antigen likely form local complexes that activate complement, generating C5a, leading to local vascular activation to recruit the antigen-primed effector T cells that mediate the CS response.

Human mast cells produce matrix metalloproteinase 9.

Extracellular matrix‐destructive enzymes, like matrix metalloproteinases (MMP), have been recognized in the process of inflammation and tissue remodeling and repair. The affected tissues often contain markedly increased numbers of mast cells. Although mast cells are capable of activating latent collagenase and proMMP, it has so far been unknown whether human mast cells themselves produce and secrete MMP9. In this study, MMP9 production by cord blood‐derived cultured human mast cells and HMC‐1 human mast cells was examined by reverse‐transcriptase PCR, gelatin zymography and Western blot analysis using an antibody against MMP9. Cultured mast cells and HMC‐1 cells treated with phorbol 12‐myristate 13‐acetate were shown to express MMP9 mRNA, and the cultured conditioned media from these cells showed gelatinolytic activity, identical with MMP9. Immunohistochemical examination was performed to detect MMP9 in tissue mast cells; mast cells localized in the skin, lung and synovial tissue showed strongly positive reactions for MMP9. Thus, these findings indicate that human mast cells can produce MMP9, which might contribute to extracellular matrix degradation and absorption in the process of allergic and nonallergic responses.

Nerve Growth Factor Activates Mast Cells Through the Collaborative Interaction with Lysophosphatidylserine Expressed on the Membrane Surface of Activated Platelets

Effect of nerve growth factor (NGF) on platelet-associated mast cell activation was investigated. Although neither NGF alone nor platelets alone induced significant 5-hydroxytriptamine (5-HT) release from rat peritoneal mast cells, marked 5-HT release was detected when costimulated with NGF and calcium ionophore-activated platelets. This response reached maximal levels as early as 5 min after the initiation of the coincubation and was completely blocked by anti-NGF Ab or by an inhibitor for a tyrosine kinase of the trkA NGF receptor. Paraformaldehyde-fixed platelets activated with either calcium ionophore or thrombin exhibited the collaborative ability, suggesting the possible involvement of some membrane molecules expressed on activated platelets in mast cell activation. Because activation of platelets induced expression of phosphatidylserine (PS) and/or lysoPS on membrane surface, and since lysoPS, unlike PS, initiated the NGF-induced 5-HT release, lysoPS expressed on activated platelets may be involved in the mast cell activation. Moreover, intradermal injection of NGF and activated platelets into the rat skin increased local vascular permeability. These findings suggested that NGF collaboratively worked with membrane lysoPS of activated platelets to induce mast cell activation. Thus, NGF released in response to inflammatory stimuli may contribute to mast cell activation in collaboration with locally activated platelets in the process of inflammations and tissue repair.

An Hour after Immunization Peritoneal B-1 Cells Are Activated to Migrate to Lymphoid Organs Where within 1 Day They Produce IgM Antibodies That Initiate Elicitation of Contact Sensitivity

Elicitation of contact sensitivity (CS), a classic example of T cell-mediated immunity, requires Ag-specific IgM Abs to trigger an initiation process. This early process leads to local recruitment of CS-effector T cells after secondary Ag challenge. These Abs are produced by the B-1 subset of B cells within 1 day after primary skin immunization. In this study we report the surprising observation that B-1 cells in the peritoneal cavity are activated as early as 1 h after naive mice are painted with a contact-sensitizing Ag on the skin of the trunk and feet to begin the initiation of CS. B-1 cells in the spleen and draining lymph nodes produce the initiating Abs by 1 day after immunization, when we found increased numbers of Ag-specific IgM Ab-producing cells in these tissues by ELISPOT assay. Importantly, we show that contact-activated peritoneal B-1 cells migrate to these lymphoid tissues and then differentiate into Ag-specific IgM Ab-producing cells, resulting in specific CS-initiating IgM Abs in the serum by 1 day. Furthermore, pertussis toxin, which is known to inhibit signaling via G protein-coupled chemokines, inhibited the migration of contact-activated peritoneal B-1 cells to the lymphoid tissues, probably due to BLR-1 (Burkitt lymphoma receptor-1). These findings indicate that within 1 h after contact skin immunization, B-1 cells in the peritoneal cavity are activated to migrate to the lymphoid tissues by chemokine-dependent mechanisms to produce serum Ag-specific IgM Abs within 1 day after immunization, leading to local recruitment of CS-effector T cells.

TLR-Dependent IL-4 Production by Invariant Vα14+Jα18+ NKT Cells to Initiate Contact Sensitivity In Vivo

LPS stimulated B-1 cell polyclonal in vivo IgM responses depend on IL-4 release by invariant Vα14+Jα18+ NKT (iNKT) cells. The IgM Abs can recruit effector T cells to mediate contact sensitivity. LPS activates the B-1 cell response just 1 day later, and depends on CD1d, iNKT cells, IL-4, TLR4, and MyD88. LPS in vivo and in vitro stimulates rapid preferential production of IL-4 in hepatic iNKT cells within 2 h. TLR4 were demonstrated in iNKT cells by flow cytometry and functional studies. Thus, innate microbial stimulation via TLR can activate iNKT cell and B-1 cell collaboration. The result is polyclonal IgM Ab responses capable of recruiting Ag-specific T cells into tissues. This may be involved in the promotion of autoimmunity by infectious agents.

Inability of IL-12 to Down-Regulate IgE Synthesis Due to Defective Production of IFN-γ in Atopic NC/Nga Mice

NC/Nga mice raised in nonsterile circumstances spontaneously suffer from atopic dermatitis-like skin lesions with IgE hyperproduction. We investigated effects of rIL-12 on the IgE production in NC/Nga mice. rIL-12 administration was successful to suppress the increase of IgE levels in BALB/c mice immunized with OVA and aluminum hydroxide, but failed to abrogate that in NC/Nga mice. Both in vivo and in vitro IFN-γ production induced by rIL-12 was less in NC/Nga mice than in BALB/c mice. Addition of rIFN-γ to rIL-4 and LPS completely abrogated IgE production by B cells of BALB/c mice, but was insufficient to suppress it by B cells of NC/Nga mice. In splenic cells pretreated with Con A, STAT4 was phosphorylated at the tyrosine residue by addition of rIL-12, which was more weakly inducible in NC/Nga mice than in BALB/c mice. Finally, we examined the preventive ability of rIL-12 on the clinical aspects of atopic dermatitis in NC/Nga mice. rIL-12 administration resulted in exacerbation of development of the skin lesions and IgE production in NC/Nga mice raised in nonsterile circumstances. These results suggest that defective production of IFN-γ by T cells less sensitive to IL-12 and low responsiveness of B cells to IFN-γ may contribute to IgE hyperproduction in NC/Nga mice, and that IL-12 may have no ability to improve the clinical aspects of NC/Nga mice.

Invariant NKT Cells Rapidly Activated via Immunization with Diverse Contact Antigens Collaborate In Vitro with B-1 Cells to Initiate Contact Sensitivity

In cutaneous contact sensitivity there is an early elicited innate cascade of complement, mast cells, and platelets activated via IgM Abs. This response is required to initiate the elicitation of acquired classical contact sensitivity by leading to local recruitment of effector T cells. We recently performed in vivo experiments showing that collaboration is required between innate-like invariant Vα14+ NKT cells (iNKT) and the innate-like B-1 B cell subset to induce this initiation process. Contact sensitization triggers iNKT cells to produce IL-4 to coactivate the B-1 cells along with specific Ag for production of the initiating IgM Abs. We now describe in vitro collaboration of iNKT and B-1 cells. Normal peritoneal B-1 cells, incubated in vitro with soluble Ag, and with 1-h in vivo immune iNKT cells producing IL-4, are activated to mediate the contact sensitivity-initiation cascade. The three components of this process can be activated by different Ag. Thus, 1-h iNKT cell activation, B-1 cell stimulation, and generation of immune effector T cells can be induced by sensitization with three different Ag to respectively generate IL-4 and Ag-specific IgM Abs, to recruit the Ag-specific effector T cells. These findings have relevance to allergic and autoimmune diseases in which infections can trigger exacerbation of T cell responses to allergens or to autoantigens.

Early delayed-type hypersensitivity eosinophil infiltrates depend on T helper 2 cytokines and interferon-γ via CXCR3 chemokines

We investigated the role of T helper (Th)1‐ and Th2‐type cytokines in delayed‐type hypersensitivity to soluble protein antigens elicited early postimmunization. Mice were sensitized by intradermal injection without adjuvants, or subcutaneously with complete Freunds adjuvant, and subsequently ear challenged intradermally. As soon as day 3, antigen‐specific eosinophil‐rich responses were elicited in wild‐type mice, but not in T‐cell receptor‐α–/– mice without adjuvant. Draining lymph node T cells stimulated with antigen secreted interleukin (IL)‐4, IL‐5 and interferon‐γ (IFN‐γ). IFN‐γ‐dependent specific immunoglobulin G (IgG)2a and IL‐4‐dependent IgG1 were also generated. Delayed‐type hypersensitivity ear swelling and local eosinophil recruitment were decreased in IL‐5–/–, IL‐4–/– and signal transducer and activator of transcription‐6 (STAT‐6)–/– mice, and with anti‐IL‐4 treatment of wild‐type mice, suggesting Th2 mechanisms. Interestingly, responses were also decreased in IFN‐γ–/– mice, and IFN‐γ protein and the IFN‐γ‐inducible CXC chemokine, IP‐10, were present in 24‐hr ear tissue extracts, suggesting Th1 effects. Finally, ear swelling, total histology and eosinophils were decreased in mice deficient in CXCR3, the chemokine receptor for IP‐10. These results suggest that both a Th2‐like (IL‐5, IL‐4 and STAT‐6) and a Th1‐like (IFN‐γ, IP‐10, CXCR3) pathway contribute to eosinophil recruitment in early delayed‐type hypersensitivity.

Interleukin-4-dependent innate collaboration between iNKT cells and B-1 B cells controls adaptative contact sensitivity

We showed that hepatic Vα14+ invariant natural killer T (iNKT) cells, via their rapid interleukin (IL)‐4 production, activate B‐1 cells to initiate contact sensitivity (CS). This innate collaboration was absent in IL‐4–/– and signal transducer and activator of transcription (STAT)‐6–/– mice and was inhibited by anti‐IL‐4 treatment. These mice have defective CS because they fail to locally recruit the sensitized effector T cells of acquired immunity. Their CS is reconstituted by transfer of downstream‐acting 1‐day immune B‐1 cells from wild‐type mice. Responses were not reconstituted with B‐1 cells from IL‐4 receptor‐α–/– or STAT‐6–/– mice, nor by IL‐4 treatment of B cell‐deficient mice at immunization. Finally, IL‐4 was preferentially and transiently produced by hepatic iNKT cells within 7 min after sensitization to mediate collaboration between innate‐like iNKT cells and the B‐1 B cells that participate in the recruitment of effector T cells in vivo.