Increased expression of Toll-like receptors by monocytes and natural killer cells in ANCA-associated vasculitis

Introduction

Toll-like receptors (TLRs) are a family of receptors that sense pathogen associated patterns such as bacterial cell wall proteins. Bacterial infections are associated with anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV). Here, we assessed the expression of TLRs 2, 4, and 9 by peripheral blood leukocytes from patients with AAV, and investigated TLR mediated responses ex vivo.

Methods

Expression of TLRs was determined in 38 AAV patients (32 remission, 6 active disease), and 20 healthy controls (HC). Membrane expression of TLRs 2, 4, and 9, and intracellular expression of TLR9 by B lymphocytes, T lymphocytes, NK cells, monocytes and granulocytes was assessed using 9-color flowcytometry. Whole blood from 13 patients and 7 HC was stimulated ex vivo with TLR 2, 4 and 9 ligands and production of cytokines was analyzed.

Results

In patients, we observed increased proportions of TLR expressing NK cells. Furthermore, patient monocytes expressed higher levels of TLR2 compared to HC, and in a subset of patients an increased proportion of TLR4⁺ monocytes was observed. Monocytes from nasal carriers of Staphylococcus aureus expressed increased levels of intracellular TLR9. Membrane expression of TLRs by B lymphocytes, T lymphocytes, and granulocytes was comparable between AAV patients and HC. Patients with active disease did not show differential TLR expression compared to patients in remission. Ex vivo responses to TLR ligands did not differ significantly between patients and HC.

Conclusions

In AAV, monocytes and NK cells display increased TLR expression. Increased TLR expression by these leukocytes, probably resulting from increased activation, could play a role in disease (re)activation.     

B lymphocytes and plasma cells express functional E-selectin by constitutive activation of NF-kappaB

E-selectin (CD62E), a cell adhesion molecule for most leukocytes, is known to be expressed exclusively on the cytokine-stimulated endothelial cells mainly by inductive activation of NF-kappaB. Using immunohistochemistry and in situ hybridization, we showed that B lymphocytes and plasma cells in the spleens and lymph nodes from nude mice (T-lymphocyte-deficient), but not from SCID mice (T- and B-lymphocyte-deficient), expressed E-selectin prior to cytokine stimulation. The expression of E-selectin was also confirmed on human B lymphocytes isolated from peripheral bloods. The mouse J774A.1 monocytes could adhere to the marginal zones of mouse spleens in an E-selectin Ab inhibitable manner, suggesting the functional activity of the expressed E-selectin. In addition, ARH-77 cells, a cell line derived from human plasma cells, were found to express E-selectin mRNA and protein and to have a NF-kappaB activity for an E-selectin promoter. NF-kappaB antagonists, such as TPCK (tosylsulfonyl phenylalanyl chloromethyl ketone), dexamethasone and a IkappaBalpha mutant plasmid could inhibit both the NF-kappaB activity and the expression of E-selectin. Transfection with an E-selectin promoter-driven reporter gene construct further verified the E-selectin promoter activity in ARH-77 cells. Again, TPCK, dexamethasone, and the IkappaBalpha mutant plasmid could neutralize this activity. These findings suggest that B lymphocytes and plasma cells can express E-selectin, which is functional for monocytic leukocytes, by a mechanism of constitutive activation of NF-kappaB.     

Cytokine regulation of IL-1 beta gene expression in the human polymorphonuclear leukocyte

Although recently polymorphonuclear leukocytes (PMN) have been identified as producers of IL-1 beta in response to LPS and granulocyte/monocyte colony stimulating factor, little is known regarding the ability of other cytokines to induce the production of IL-1 beta in the PMN. Inasmuch as IL-1 and TNF have been shown to be important priming agents, as well as agents that induce migration of PMN, we investigated their effect on IL-1 beta gene expression in human peripheral blood PMN. In the present study, we demonstrate that human peripheral blood PMN produce IL-1 beta in response to IL-1 alpha, IL-1 beta, and TNF-alpha. Control (unstimulated) human PMN had virtually undetectable levels of IL-1 beta mRNA. Either IL-1 beta or TNF, induced PMN to transiently express IL-1 beta mRNA with peak expression at 1 h, returning to untreated levels by 2 h. A dose response indicated that as little as 0.05 ng/ml of IL-1 beta or TNF resulted in IL-1 beta induction, with maximal effects at 1 ng/ml of IL-1 beta and 5 ng/ml of TNF. IL-1 alpha or IL-1 beta exhibited similar dose responses in IL-1 beta mRNA induction. Inasmuch as cytokines have been shown to have synergistic effects in cell function studies, we induced PMN with a combination of maximally effective doses of TNF plus IL-1 beta. They demonstrated a cooperative effect on IL-1 beta gene expression, in that mRNA levels were sustained for three hours. IL-1 beta Ag expression, as measured by ELISA, paralleled IL-1 beta mRNA expression with cell associated peak levels at 2 to 4 h. IL-1 beta Ag levels in PMN lysates and supernatants correlated with IL-1 beta mRNA levels, i.e., TNF + IL-1 greater than TNF greater than IL-1. Thus, these studies represent the first demonstration of IL-1 and TNF induction of IL-1 beta gene expression in the PMN. Furthermore, the time course of induction is unique to the PMN, with peak induction of mRNA at 1 h, which is consistent with the short lived nature of these cells in inflammatory lesions.     

Target cells for interferon production in human leukocytes stimulated by sendai virus.

Whole leukocytes, mononuclear cells, polymorphonuclear cells (PMN), MONOCYTES, PURIFIED LYMPHOCYTES, AND T (rosette-forming cells, RFC) and non-T (nonrosette-forming cells, nonRFC) lymphocytes isolated from the human peripheral blood were stimulated by Sendai virus, respectively, and examined for interferon production in their culture fluids. High levels of interferon were produced by mononuclear cells, but not by PMN. Removal of monocytes from the mononuclear cell population did not affect at all the levels of interferon produced, although it strongly suppressed interferon induction by polyinosinic-polycytidylic acid (poly IC) and mitogenic response to phytohemagglutinin (PHA) of the lymphocytes. Purified monocytes and T lymphocytes were unresponsive to the virus. In contrast, a population of purified non-T lymphocytes produced high levels of interferon. Addition of monocytes to the interferon-producing non-T lymphocytes did not affect the levels of interferon produced. No detectable levels of interferon were produced in the mixture of T lymphocytes and monocytes. It is concluded that non-T lymphocytes may be a major target for interferon induction of human leukocytes by Sendai virus.     

Expression of chemokine-like factor 1 is upregulated during T lymphocyte activation

Chemokine-like factor 1 (CKLF1) is a cytokine with chemotactic effects on leukocytes and a functional ligand of CCR4. This cytokine is widely expressed and the level of expression is reported to be upregulated in asthma and rheumatoid arthritis (RA), disease conditions in which T lymphocytes are over-activated. In order to determine the expression profile of CKLF1 in activated T lymphocytes, we first employed a PCR-based method on human blood fractions cDNA panels and found that CKLF1 was upregulated in activated CD4+ and CD8+ cells, with no obvious changes in CD19+ cells. We further performed kinetic analyses of CKLF1 expression in phytohemagglutinin (PHA)-stimulated human peripheral blood lymphocytes (PBL) at both the mRNA and protein levels. In resting PBL, the constitutive expression of CKLF1 was low at mRNA level and barely detectable at the protein level; however, both were remarkably upregulated by PHA, appearing at 8h after PHA-stimulation and persisting up to 72h. These results suggest that CKLF1 may be involved in T lymphocyte activation and further study of CKLF1 function will prove valuable.     

Leptin indirectly activates human neutrophils via induction of TNF-alpha

Leptin, the satiety hormone, appears to act as a link between nutritional status and immune function. It has been shown to elicit a number of immunoregulatory effects, including the promotion of T cell proliferative responses, and the induction of proinflammatory cytokines. Leptin deficiency is associated with an increased susceptibility to infection. As polymorphonuclear neutrophils (PMN) play a major role in innate immunity and host defense against infection, this study evaluated the influence of leptin on PMN activation. The presence of leptin receptor in human PMN was determined both at mRNA and protein levels, and the effect of leptin on PMN activation, as assessed by CD11b expression, was evaluated using flow cytometry. In contrast to monocytes, which express both the short and long forms of the leptin receptor (Ob-Ra and Ob-Rb, respectively), PMN expressed only Ob-Ra. Leptin up-regulated the expression of CD11b, an early marker of PMN activation, on PMN in whole blood, yet it had no effect on purified PMN, even those treated by submaximal doses of TNF-alpha or PMA. The kinetics of leptin-induced activation in whole blood were consistent with an indirect effect mediated by monocytes, and 71% of the leptin-stimulatory effect on PMN was blocked by a TNF-alpha inhibitor. Leptin-mediated induction of CD11b expression was observed when purified PMN were coincubated with purified monocytes. In conclusion, although leptin activates PMN, it does so indirectly via TNF-alpha release from monocytes. These findings provide an additional link among the obesity-derived hormone leptin, innate immune function, and infectious disease.     

Granulocyte-macrophage colony stimulating factor up-regulates CCR1 in human neutrophils

Neutrophils (polymorphonuclear leukocytes; PMN) are phagocytic cells instrumental in the clearance of infectious pathogens. Human PMN are commonly thought to respond primarily to chemokines from the CXC family. However, recent findings suggest that under specific cytokine activation conditions, PMN can also respond to some CC chemokines. In this study, the effect of GM-CSF, a well-characterized PMN priming and maturation factor, on CC-chemokine receptor (CCR) expression in PMN was investigated. Constitutive expression of CCR1 and CCR3 mRNA in PMN was detected by ribonuclease protection assay. Following incubation of PMN with GM-CSF (0.01-10 ng/ml; 6 h) CCR1 mRNA expression was rapidly (approximately 1 h) up-regulated. In contrast, no significant induction of CCR2, CCR3, CCR4, or CCR5 mRNA was observed. CCR1 protein was also up-regulated by GM-CSF stimulation. GM-CSF-induced up-regulation of CCR1 showed functional consequences because GM-CSF-treated PMN, but not control cells, responded to the CC chemokines macrophage inflammatory protein-1alpha, monocyte chemoattractant protein-3, and RANTES in assays of chemotactic migration and intracellular calcium mobilization. These results suggest that PMN activated by the proinflammatory cytokine GM-CSF can change their receptor expression pattern and become responsive to CC chemokines.