AMD3465, a novel CXCR4 receptor antagonist, abrogates schistosomal antigen-elicited (type-2) pulmonary granuloma formation

CXCR4 is a major receptor for CXCL12 and is known to participate in multiple physiological systems. The present study tested a second generation CXCR4 antagonist, AMD3465, for effects on highly defined models of Th1- and Th2-cell-mediated hypersensitivity-type pulmonary granuloma formation. Type-1 and type-2 granulomas were induced, respectively, by intravenous challenge of sensitized CBA/J mice with Mycobacteria bovis purified protein derivative- or Schistosoma mansoni egg antigen-coated beads. Before challenge, mice were implanted with osmotic pumps releasing AMD3465 at 5 microg/hour (6 mg/kg/day). Compared to vehicle, AMD3465 had minimal effect on type-1 inflammation or cytokine responses in draining lymph nodes, but the type-2 inflammation was significantly abrogated with reductions in lesion size and eosinophil content as well as abrogated interleukin (IL)-5, IL-10, and IL-13 cytokine production in draining lymph nodes. The biased effect of AMD3465 correlated with greater CXCR4 ligand expression in the type-2 model. Treatment during a primary response impaired lymph node IL-2 production after both Mycobacteria bovis purified protein derivative and Schistosoma mansoni egg antigen challenge indicating an unbiased effect during immune induction. In summary, CXCR4 blockade inhibited eosinophil recruitment during type-2 granuloma formation and interfered with primary and secondary T-cell activation events in lymphoid tissue, suggesting potential therapeutic application for chronic hypersensitivity diseases.     

Frequency and phenotype of natural killer cells and natural killer cell subsets in bovine lymphoid compartments and blood

Natural killer (NK) cells are widely distributed in lymphoid and non‐lymphoid tissues, but little is known about the recirculation of NK cells between blood and tissues. This is relevant to understanding recirculation in the steady‐state and also for determining the roles for NK cells in vaccine‐induced immunity and responses to infection. Therefore, the percentage of NK cells and their phenotype across peripheral blood, afferent lymph and lymph nodes in steady‐state conditions was investigated in cattle using the pseudo‐afferent lymphatic cannulation model. CD2⁺ CD25^lo NK cells were the predominant subset of NK cells within the blood. In contrast, CD2⁻ CD25^hi NK cells were the main subset present within the skin‐draining afferent lymphatic vessels and lymph nodes, indicating that CD2⁻ NK cells are the principal NK cell subset trafficking to lymph nodes via the afferent lymphatic vessel. Furthermore, a low percentage of NK cells were present in efferent lymph, which were predominantly of the CD2⁻ subset, indicating that NK cells can egress from lymph nodes and return to circulation in steady‐state conditions. These compartmentalization data indicate that NK cells represent a population of recirculating lymphocytes in steady‐state conditions and therefore may be important during immune responses to vaccination or infection.     

Modification of caecal lymphoid tissue and relationship to granuloma formation in sporadic ileocaecal tuberculosis

The histopathology of eight cases of sporadic ileocaecal tuberculosis is described with particular reference to caecal lymphoid tissue. Adjacent to areas of ulceration in all cases there was an increase in lymphoglandular complexes (LGC) and proliferation of paravascular lymphoid aggregates deeper in the gut wall. Early and fully-developed granulomas were present in locations comparable to LGC and lymphoid aggregates. Immunocytochemical staining of paraffin sections with monoclonal antibodies UCHL1 (T-lymphocyte membrane antigen) and LN-1, LN-2 and LN-3 (B-lymphocyte and Ia antigens) showed that central cells in LGC and lymphoid aggregates stained like follicular centre B-lymphocytes. Both LGC and lymphoid aggregates had a distinct peripheral rim of cells staining as T-lymphocytes, but LN-2 and LN-3 also stained scattered peripheral cells, some of which were recognizable as interdigitating reticulum cells. Most lymphocytes within and around granulomas stained as T-lymphocytes. In lymph nodes, granulomas appeared to occur first at the periphery of, and later to efface, cortical follicles. Lymph node compartments showed the expected T- and B-zonation, and lymphocytes associated with granulomas stained as in caecal granulomas. Our observations suggest that LGC are sites of mycobacterial antigen sampling, of T-lymphocyte and macrophage activation, and of (potential) granuloma formation in ileocaecal tuberculosis. Lymphoid aggregates deeper in the gut wall probably subserve a similar function during extension of the lesion. The location of both LGC and lymphoid aggregates beside lymphatics is suited to the transfer of their cellular constituents throughout the gut and to regional lymph nodes.     

Differences in activation and tissue homing markers of natural killer cell subsets during acute dengue infection

Dengue virus (DENV) infection is considered one of the most important mosquito‐borne diseases. It causes a spectrum of illness that could be due to qualitative and/or quantitative difference(s) of the natural killer (NK) cell responses during acute DENV infection. This view prompted us to perform a detailed phenotypic comparative characterization of NK cell subsets from DENV‐infected patients with dengue fever (DF), patients with dengue haemorrhagic fever (DHF) and healthy controls. The activation/differentiation molecules, CD69 and CD57 and a variety of tissue homing molecules were analysed on the CD56^hi CD16^? and CD56^lo CD16⁺ NK cells. Although there was no increase in the frequency of the total NK cells during DENV infection compared with the healthy individuals, there was a significant increase in the frequency of the CD56^hi CD16^? subset and the frequency of CD69 expression by both NK cell subsets during the febrile phase of infection. We also found an increase in the frequencies of cells expressing CD69 and CD57 in the CD56^lo CD16⁺ subset compared with those in the CD56^hi CD16^? subset. Moreover, although the CD56^lo CD16⁺ subset contained a high frequency of cells expressing skin‐homing markers, the CD56^hi CD16^? subset contained a high frequency of cells expressing bone marrow and lymph node trafficking markers. Interestingly, no differences of these NK cell subsets were noted in samples from patients with DF versus those with DHF. These findings suggest that activation and differentiation and the patterns of tissue homing molecules of the two major NK cell subsets are different and that these might play a critical role in the immune response against acute DENV infection.     

Human natural killer cell receptor functions and their implication in diseases

Both active and passive anti-β-amyloid (Aβ) immunotherapies for the treatment of Alzheimer's disease (AD) have demonstrated clearance of brain Aβ deposits. Among passive immunotherapeutics, two Phase III clinical trials in mild-to-moderate AD patients with bapineuzumab, a humanized monoclonal antibody directed at the N-terminal sequence of Aβ, were disappointing. Also solanezumab, directed at the mid-region of Aβ, failed in two Phase III trials in mild-to-moderate AD. Another Phase III trial with solanezumab is ongoing in mildly affected AD patients based on encouraging results in this subgroup. Second-generation active Aβ vaccines (CAD106, ACC-001, and Affitope AD02) and new passive anti-Aβ immunotherapies (gantenerumab and crenezumab) have been developed and are under clinical testing. These new anti-Aβ immunotherapies are being tested in prodromal AD, in presymptomatic subjects with AD-related mutations, or in asymptomatic subjects at risk of developing AD. These primary and secondary prevention trials will definitely test the Aβ cascade hypothesis of AD.     

Sphingosine-1-phosphate receptor type-1 agonism impairs blood dendritic cell chemotaxis and skin dendritic cell migration to lymph nodes under inflammatory conditions

SEW2871 is a potent sphingosine-1-phosphate receptor type-1 (S1P(1))-selective agonist that induces peripheral lymphopenia through sequestration of lymphocytes into secondary lymphoid organs, similar to the non-selective sphingosine-1-phosphate (S1P) receptor agonist FTY720. FTY720 has been reported to interfere with human dendritic cell (DC) effector functions and both FTY720 and SEW2871 have been shown to modulate murine DC trafficking in vivo. Little is known about the possible effects of SEW2871 on human and murine DC functions. Here, we demonstrate that in contrast to FTY720, SEW2871 does not induce down-regulation of S1P(1) in human DCs and thus does not exert a functional antagonism at S1P(1). Notably, the compound was found to impair chemotaxis of immature and mature human DCs in vitro, possibly by interfering with the activation of p44/p42 and p38 mitogen-activated protein kinase signaling pathways. Comparative FACS analyses show that SEW2871 mediates CD18 down-regulation on mature human DCs. The influence on DC migration could be confirmed with in vivo assays using BALB/c mice in which SEW2871 impairs the migration of CD11c+ DC and CD207+ Langerhans cells (LC) to the draining lymph nodes (LNs) under inflammatory conditions. These results suggest that the S1P-S1P(1) axis might not only control lymphocyte trafficking but also play a pivotal role in DC migration from the skin to LN.     

Early appearance and activation of natural killer cells in tumor-infiltrating lymphoid cells during tumor development

We investigated NK cell infiltration into tumor developing lesions at early stage of tumor development after intraperitoneal inoculation of 3LL lung carcinoma into syngeneic C57BL/6 mice. Natural killer (NK) cells, which were detected by anti-NK 1.1 monoclonal antibody (mAb), remarkably increased in number in tumor-developing lesions (peritoneal cavity) as early as day 3 after inoculation of 3LL. The tumor-infiltrating NK cells from 3LL-inoculated mice produced a high level of interferon-γ by co-culture with 3LL and showed enhanced cytotoxic activities against both NK-sensitive (YAC-1) and NK-resistant (3LL and P815) tumors. Furthermore, mice depleted of NK cells by injection of anti-NK 1.1 mAb or antiasialo GM1 antibody showed shorter survival times after intraperitoneal inoculation of 3LL when compared with control mice. These results suggest that NK cells infiltrate the tumor-developing lesion at an early stage and may participate in the early protection against tumors through production of a high amount of interferon-γ and enhanced cytotoxicity at tumor-bearing sites.     

Distribution of Toll-like receptor 1 and Toll-like receptor 2 in human lymphoid tissue

To determine how distinct receptors of the immune system can contribute to innate immunity, we investigated the pattern of Toll-like receptor 1 (TLR1) and TLR2 expression in human lymphoid tissue. We found that TLR1 and TLR2 were co-expressed on cells of the innate immune system, including macrophages and dendritic cells. In addition, TLR1 and TLR2 were expressed in mucosa-associated lymphoid tissue on tonsillar crypt epithelium. Of the lymphoid tissue examined, spleen expressed the highest levels of TLR2. Although TLR1- and TLR2-positive cells were in close proximity to T lymphocytes in vivo, lymphocytes themselves were devoid of TLR1 and TLR2 expression. The co-expression of TLR1 and TLR2 on myeloid cells in lymphoid tissue provides the host with the ability to respond to a variety of microbial ligands at sites conducive to the generation of an immune response.     

Lethal influenza infection in the absence of the natural killer cell receptor gene Ncr1

The elimination of viruses and tumors by natural killer cells is mediated by specific natural killer cell receptors. To study the in vivo function of a principal activating natural killer cell receptor, NCR1 (NKp46 in humans), we replaced the gene encoding this receptor (Ncr1) with a green fluorescent protein reporter cassette. There was enhanced spread of certain tumors in 129/Sv but not C57BL/6 Ncr1(gfp/gfp) mice, and influenza virus infection was lethal in both 129/Sv and C57BL/6 Ncr1(gfp/gfp) mice. We noted accumulation of natural killer cells at the site of influenza infection by tracking the green fluorescent protein. Our results demonstrate a critical function for Ncr1 in the in vivo eradication of influenza virus.     

Cytokine responses during mycobacterial and schistosomal antigen-induced pulmonary granuloma formation. Production of Th1 and Th2 cytokines and relative contribution of tumor necrosis factor.

Synchronized pulmonary granulomas (GRs) were induced in presensitized mice by intravenous embolization of polymer beads bound with purified protein derivative (PPD) of Mycobacteria tuberculosis or soluble antigens derived from Schistosoma mansoni eggs (SEA). Uncoated beads served as a foreign body control (CON). Antigen-coated beads elicited GRs with characteristic epithelioid macrophages and multinucleate giant cells by 4 days after embolization. Unlike PPD GR, SEA bead lesions contained eosinophils, whereas CON beads elicited only a limited mononuclear infiltrate. GRs and draining lymph nodes (LN) were assessed on days 2, 4, and 8 for Th1-(interleukin-2 [IL-2], interferon-gamma[IFN] and Th2-type (IL-4, IL-5, and IL-10) cytokines. CON GR produced only a small amount of IFN-gamma on day 2 and failed to induce a significant response in draining LN. In contrast, both PPD and SEA antigen-coated beads induced reactive lymphoid hyperplasia but differed greatly in local and regional cytokine profiles. PPD GR produced IFN-gamma on day 2 and the draining LN produced predominantly Th1 cytokines on days 2 and 4. In contrast, SEA beads GRs were dominated by Th2 cytokines. The corresponding LN produced IL-2 and IL-4 on day 2; IL-2, IL-4, IFN-gamma, and IL-10 on day 4; then IL-2, IFN-gamma, and IL-4 on day 8, probably reflecting maturational changes of T cells. Macrophages (MP) from bead GR also showed different patterns of IL-6 and tumor necrosis factor (TNF) production. Compared with CON GR, MPs from PPD GR were weak sources of IL-6, whereas those of SEA GR showed enhanced and accelerated production. In contrast, MP of PPD GR had augmented TNF-producing capacity, whereas those of SEA GR showed delayed TNF production. In vivo depletion of TNF, respectively, caused 40 and 10% decreases in PPD GR and SEA GR but had no effect on CON GR area, indicating that TNF contributed to a greater degree to the PPD response. These data show that depending on the inciting agent, GR can be mediated by different cytokines. Characterization of inflammatory lesions by cytokine profiles should allow design of more rational therapeutic interventions.     

TIA-1 expression in lymphoid neoplasms. Identification of subsets with cytotoxic T lymphocyte or natural killer cell differentiation.

TIA-I is a 15-kd cytotoxic granule-associated protein expressed in natural killer (NK) cells and cytotoxic T lymphocytes. TIA-1 expression was evaluated by paraffin immunohistochemistry in 115 T- or NK-cell neoplasms, 45 B-cell neoplasms, and 45 Hodgkin''s lymphomas. TIA-1-positive granules were identified within the cytoplasm of neoplastic cells in 6/6 large granular lymphocytic leukemias, 11/11 hepatosplenic T-cell lymphomas, 15/15 intestinal T-cell lymphomas, 6/6 NK-like T-cell lymphomas of no special type, 2/2 NK-cell lymphomas, 8/9 nasal T/NK-cell lymphomas, 7/8 subcutaneous T-cell lymphomas, 4/5 pulmonary T- or NK-cell angiocentric lymphomas (lymphomatoid granulomatosis), 12/19 T-cell anaplastic large-cell lymphomas, 2/12 nodal peripheral T-cell lymphomas, 1/3 CD8+ cutaneous T-cell lymphomas, and 5/38 classical Hodgkin''s disease. All B-cell neoplasms, nodular lymphocyte-predominant Hodgkin''s disease (7 cases), CD4+ cutaneous T-cell lymphomas (6 cases), adult T-cell leukemia/lymphomas (3 cases), T-cell chronic or prolymphocytic leukemias (3 cases), and T-cell lymphoblastic leukemia/lymphomas (7-cases) were TIA-1 negative. These findings indicate that most large granular lymphocytic leukemias, hepatosplenic T-cell lymphomas, intestinal T-cell lymphomas, NK-like T-cell lymphomas, NK-cell lymphomas, nasal T/NK-cell lymphomas, subcutaneous T-cell lymphomas, pulmonary angiocentric lymphomas of T or NK phenotype, and anaplastic large-cell lymphomas are cytotoxic T-or NK-cell neoplasms.     

Differences between squamous cell carcinoma and keratoacanthoma in angiotensin type-1 receptor expression

Angiotensin II receptors are the specific receptors of angiotensin II of the renin-angiotensin system. The existence and role of the receptors in the skin have not been determined. We immunohistochemically studied the expression of angiotensin receptors in the human skin. The results demonstrated the expression of angiotensin type 1 receptor (AT1) in the normal human suprabasal epidermis. The expression pattern suggests the role of AT1 in association with differentiation. In addition, we studied the expression of AT1 in squamous cell carcinoma (SCC) of the skin, SCC of the lip, and keratoacanthoma (KA). Our experiments showed that high, intermediate, and low levels of AT1 were observed in 37 (74.0%), 7 (14.0%), and 2 (4.0%) of 50 cases of SCC of the skin, respectively, and the negative periphery pattern was observed in 17 (77.3%) of 22 cases of KA. These observations suggest that the immunohistochemical study of AT1 is useful to distinguish SCC from KA. Studying the role and distribution of AT1 may help in understanding the pathophysiology of the skin.     

Critical roles of co‐activation receptor DNAX accessory molecule‐1 in natural killer cell immunity

Natural killer (NK) cells, which can exert early and powerful anti‐tumour and anti‐viral responses, are important components of the innate immune system. DNAX accessory molecule‐1 (DNAM‐1) is an activating receptor molecule expressed on the surface of NK cells. Recent findings suggest that DNAM‐1 is a critical regulator of NK cell biology. DNAM‐1 is involved in NK cell education and differentiation, and also plays a pivotal role in the development of cancer, viral infections and immune‐related diseases. However, tumours and viruses have developed multiple mechanisms to evade the immune system. They are able to impair DNAM‐1 activity by targeting the DNAM‐1 receptor–ligand system. We have reviewed the roles of DNAM‐1, and its biological functions, with respect to NK cell biology and DNAM‐1 chimeric antigen receptor‐based immunotherapy.     

A sphingosine-1-phosphate receptor 1 agonist inhibits tertiary lymphoid tissue reactivation and hypersensitivity in the lung

Hypersensitivity pneumonitis is characterized by pulmonary accumulation of B-cell-rich tertiary lymphoid tissues (TLTs), which are alleged sites of amplification for antigen-specific responses. The sphingosine-1-phosphate receptor 1 (S1P₁) regulates key mechanisms underlying lymphoid tissue biology and its chemical modulation causes lymphocyte retention in lymph nodes. Given the putative immunopathogenic impact of lymphocyte accumulation in TLTs, we investigated whether or not chemical modulation of S1P₁ caused lymphocyte retention within TLTs in a model of hypersensitivity pneumonitis. Mice were exposed subchronically to Methanosphaera stadtmanae (MSS) in order to induce an hypersensitivity pneumonitis-like disease. MSS exposure induced B-cell-rich TLTs surrounded by S1P₁-positive microvessels. Upon MSS rechallenge, the S1P₁ agonist RP001 prevented the pulmonary increase of CXCL13, a chief regulator of B-cell recruitment in lymphoid tissues. This was associated with a complete inhibition of MSS rechallenge-induced TLT enlargement and with a 2.3-fold reduction of MSS-specific antibody titers in the lung. Interference with TLT reactivation was associated with a 77% reduction of neutrophil accumulation and with full inhibition of protein-rich leakage in the airways. Thus, an S1P₁ agonist hinders TLT enlargement upon antigenic rechallenge and inhibits key pathognomonic features of experimental hypersensitivity pneumonitis.     

Family study of natural killer cell activity in C1q-deficient patients with systemic lupus erythematosus-like syndrome: association between impaired natural killer cell function and C1q deficiency

Impaired natural killer (NK) cell activity has been found in patients with systemic lupus erythematosus (SLE)-like syndrome. The mechanism by which NK cell function is impaired in SLE patients is not quite clear. We report here a family study of NK cell activity in C1q-deficient patients with SLE-like syndrome. In both SLE-active and SLE-inactive stages of the disease, NK cell function was significantly impaired when compared with the healthy controls (10.6 +/- 2.3% and 16.9 +/- 4.8% to 34.7 +/- 9.6%, p less than 0.025). On the other hand, differences in NK cell cytotoxicity between SLE-active and SLE-inactive members of the family were not statistically relevant (p less than 0.1). Further, we found no correlation between NK cell activity and clinical or laboratory values, except for a positive correlation between function of NK cells and C1q and CH50 values, respectively (rs = 0.93, 0.01 less than p less than 0.02). To our knowledge, this is the first report on a notable association between impaired NK cell activity and C1q deficiency. The type of inheritance of C1q deficiency in this family is also discussed.     

Role of angiotensin II and angiotensin type-1 receptor in scorpion venom-induced cardiac and aortic tissue inflammation

Scorpion stings are mainly associated with cardiovascular disturbances that may be the cause of death. In this study, the involvement of angiotensin II (Ang II) in cardiac and aortic inflammatory response was studied. Mice were injected with Androctonus australis hector (Aah) scorpion venom (0.5 mg/kg, subcutaneously), in the presence or absence of an angiotensin converting enzyme (ACE) inhibitor, captopril (15 mg/kg/day/1 day intraperitoneally) or an angiotensin type-1 receptor (AT1R) antagonist, valsartan (15 mg/kg/day/15 days, orally). In the envenomed group, results revealed severe tissue alterations with a concomitant increase of metabolic enzymes (CK and CK-MB) in sera. An important inflammatory cell (neutrophil and eosinophil) infiltration into the heart and aorta were observed, accompanied by imbalanced redox status (NO, MDA, catalase and GSH) and high cytokine levels (IL-6 and TNF-α) in sera with the expression of MMP-2 and MMP-9 metalloproteinases. However, the blockade of the actions of AngII by the ACE inhibitor or by the AT1R antagonist prevented cardiac and aortic tissue alterations, inflammatory cell infiltration, as well as the oxidative stress generation and cytokine and metalloproteinase expression. These results suggest the involvement of AngII, through its AT1R in the inflammation induced by Aah venom, in the heart and the aorta.     

Immunohistochemical demonstration of interleukin-1 receptor antagonist protein and interleukin-1 in human lymphoid tissue and granulomas.

Human interleukin 1 receptor antagonist protein (IRAP) is a specific antagonist of interleukin 1 (IL-1) action in both in vitro and in vivo experimental models. Presently, the significance of this protein in human pathophysiology is unknown. In the present study, monoclonal antibodies against IRAP were prepared and used to demonstrate IRAP expression in human tissues, immunohistochemically. Specifically, this study focused on lymphoid tissues and granulomatous inflammatory reactions since IL-1 is believed to play roles in lymphocyte development and inflammation. In addition, these tissues were also stained for IL-1 beta to compare the expression of agonist and antagonist. These findings indicate that IRAP expression is largely limited to macrophages and their derivatives. Strong IRAP expression was observed in germinal center macrophages of lymph nodes, spleen, and tonsil. In contrast, IL-1 was marginally expressed in these organs. Granulomas associated with active M. tuberculosis infection, sarcoidosis and foreign bodies all contained strongly IRAP positive cells, which included macrophages, epithelioid cells and multinucleate giant cells. Unlike reactive lymphoid tissue, tuberculous and sarcoid granulomas also contained IL-1 positive cells which included macrophages and their derivatives, as well as some stromal cells. Foreign body lesions showed minimal IL-1 expression. Interestingly, granulomas in patients with acquired immunodeficiency disease (AIDS) associated with M. avium-intracellulare contained IRAP positive cells but were negative for IL-1 expression. Taken together, these findings suggest that IRAP takes part in both physiologic and pathologic reactions. Moreover, they provide a basis to design future studies to determine the precise contribution of IRAP to these reactions.