The role of cytotoxicity in lymphocyte homeostasis.

Several human inherited immune disorders lead to the same fatal lymphoproliferative syndrome, called the hemophagocytic syndrome. Through defective perforin expression or transport, these disorders highlight the determinant role of the secretory cytotoxic pathway in the regulation of the immune response and in lymphocyte homeostasis. In addition, new effectors of this secretory pathway have been identified.     

Tumour suppressor Fus1 provides a molecular link between inflammatory response and mitochondrial homeostasis

Fus1, encoded by a 3p21.3 tumour suppressor gene, is down-regulated, mutated or lost in the majority of inflammatory thoracic malignancies. The mitochondrial localization of Fus1 stimulated us to investigate how Fus1 modulates inflammatory response and mitochondrial function in a mouse model of asbestos-induced peritoneal inflammation. Asbestos treatment resulted in a decreased Fus1 expression in wild-type (WT) peritoneal immune cells, suggesting that asbestos exposure may compromise the Fus1-mediated inflammatory response. Untreated Fus1(-/-) mice had an ~eight-fold higher proportion of peritoneal granulocytes than Fus1(+/+) mice, pointing at ongoing chronic inflammation. Fus1(-/-) mice exhibited a perturbed inflammatory response to asbestos, reflected in decreased immune organ weight and peritoneal fluid protein concentration, along with an increased proportion of peritoneal macrophages. Fus1(-/-) immune cells showed augmented asbestos-induced activation of key inflammatory, anti-oxidant and genotoxic stress response proteins ERK1/2, NFκB, SOD2, γH2AX, etc. Moreover, Fus1(-/-) mice demonstrated altered dynamics of pro- and anti-inflammatory cytokine expression, such as IFNγ, TNFα, IL-1A, IL-1B and IL-10. 'Late' response cytokine Ccl5 was persistently under-expressed in Fus1(-/-) immune cells at both basal and asbestos-activated states. We observed an asbestos-related difference in the size of CD3(+) CD4(-) CD8(-) DN T cell subset that was expanded four-fold in Fus1(-/-) mice. Finally, we demonstrated Fus1-dependent basal and asbestos-induced changes in major mitochondrial parameters (ROS production, mitochondrial potential and UCP2 expression) in Fus1(-/-) immune cells and in Fus1-depleted cancer cells, thus supporting our hypothesis that Fus1 establishes its immune- and tumour-suppressive activities via regulation of mitochondrial homeostasis.     

A microcytotoxicity assay for thyroid-specific cytotoxic antibody, antibody-dependent cell-mediated cytotoxicity and direct lymphocyte cytotoxicity using human thyroid cells

A solid-phase enzyme immunoassay for the detection of antibodies, specific for hemoglobin (Hb) is described. The application of glutaraldehyde resulted in a sensitive assay and allowed the use of urea, which is an important advantage if polypeptides not soluble in aqueous buffers are to be used. Mutation-carrying Hb chains can be purified, solubilized in urea and used in the immunoassay to monitor the purification and selection of antibodies specific for these variants. Specific antibodies are the main tools for the development of a hemoglobin-locus mutation system for detection of potentially mutagenic environmental agents. With erythrocytes as target cells, this system permits in vivo monitoring of subjects under exposure. Conventional antibody production, however, frequently turns out to be unsuccessful. The production of monoclonal antibodies has several advantages over conventional antibody production, but a sensitive antibody screening system is essential. Because of the sensitivity and the ease with which a large panel of antibody fractions against a vast panel of Hb antigens can be examined, the described immunoassay has potential value for the screening of hybridoma cultures.     

The contribution of autophagy to lymphocyte survival and homeostasis

Summary: Over the life span of a T lymphocyte, from thymic development to death, it is subjected to a variety of stresses and stimuli. Upon receipt of each stress or stimulus, a potentially life-changing fate decision must be made, namely, whether to commit to a form of programmed cell death or to make the necessary adaptations to effectively deal with the changing environment. In our laboratory, we have identified several stresses that a T lymphocyte will encounter during a normal life span. Our studies have focused on how T cells utilize autophagy to get a grasp on the situation, or in cases in which survival is untenable, how T cells use autophagy to hasten their demise. This review focuses on the functions of T-cell autophagy in maintaining homeostasis, eliminating excess or dangerous levels of mitochondria, trimming levels of endoplasmic reticulum, and promoting a healthy metabolic level to allow cells to perform as productive components of the immune system. In addition, the use of autophagy signaling molecules to perform autophagy-independent tasks involved in the maintenance of immune homeostasis is discussed.     

Primary mixed lymphocyte responses to HLA-DP

Combinations of peripheral blood lymphocytes, matched or mismatched for HLA-DP, were analyzed in primary one-way mixed lymphocyte culture experiments. Proliferative responses as correlated with tritiated thymidine uptake were assessed over a kinetic range of 5-15 days. A proliferative response was observed between DP-mismatched combinations, whereas combinations matched for DP and all other HLA alloantigens did not elicit significant proliferation. Optimal responses were observed 9 days after the combination of 1 x 10(5) responder and stimulator cells. Responses were blocked by anti-DP monoclonal antibodies. These studies demonstrate the complexity of the primary mixed lymphocyte culture system and suggest that DP alloantigens should be considered when anomalous responses are obtained.     

Spontaneous and lectin-dependent cellular cytotoxicity by lymphocyte subpopulations against cell lines susceptible or resistant to spontaneous cytotoxicity

In the present study we have investigated whether cell lines, which are resistant to spontaneous cytotoxicity, can be killed by lectin–dependent cellular cytotoxicity (LDCC), the expression of EAC (7S) receptors on LDCC effector cells, and the relationship between spontaneous cytotoxicity and LDCC when EAC (7S) receptor–positive or –negative subpopulations are tested against different target cell lines.     

LKB1 interacts with and phosphorylates PTEN: a functional link between two proteins involved in cancer predisposing syndromes

Germline mutations of the LKB1 (STK11) tumor suppressor genelead to Peutz-Jeghers syndrome (PJS) and predisposition to cancer.LKB1 encodes a serine/threonine kinase generally inactivatedin PJS patients. We identified the dual phosphatase and tumorsuppressor protein PTEN as an LKB1-interacting protein. SeveralLKB1 point mutations associated with PJS disrupt the interactionwith PTEN suggesting that the loss of this interaction mightcontribute to PJS. Although PTEN and LKB1 are predominantlycytoplasmic and nuclear, respectively, their interaction leadsto a cytoplasmic relocalization of LKB1. In addition, we showthat PTEN is a substrate of the kinase LKB1 in vitro. As PTENis a dual phosphatase mutated in autosomal inherited disorderswith phenotypes similar to those of PJS (Bannayan–Riley–Ruvalcabasyndrome and Cowden disease), our study suggests a functionallink between the proteins involved in different hamartomatouspolyposis syndromes and emphasizes the central role played byLKB1 as a tumor suppressor in the small intestine.     

CD83 regulates lymphocyte maturation, activation and homeostasis

The transmembrane CD83 molecule, a conserved member of the immunoglobulin superfamily, is known as one of the most characteristic cell surface markers for fully matured dendritic cells (DCs) in the peripheral circulation. An essential role for CD83 on murine DCs has not been found; however, evidence shows that its function primarily lies in the regulation of T- and B-lymphocyte maturation and in the regulation of their peripheral responses. Here, we review evidence for a role of CD83 in central lymphocyte maturation and novel, sometimes contradictory findings, regarding the function of CD83 in peripheral immune responses.     

Altered B lymphocyte homeostasis and functions in systemic sclerosis

Beyond the production of autoantibodies, B-cells are thought to play a role in systemic sclerosis (SSc) by secreting proinflammatory/profibrotic cytokines. B-cells are a heterogeneous population with different subsets distinguished by their phenotypes and cytokine production. Data about B-cell subsets, cytokine production and intracellular pathways leading to this production are scarce in SSc. The aim of our study was to describe B-cell homeostasis, activation, proliferation, cytokine production in B-cells and serum and B-cell intracellular signaling pathways in SSc. We hypothezided that B-cell homeostasis and cytokine production were altered in SSc and could be explained by serum cytokine as well as by intracellular signaling pathway abnormalities.Forty SSc patients and 20 healthy controls (HC) were prospectively included. B-cell subsets were determined by flow cytometry using CD19, CD21, CD24, CD38, CD27, IgM and IgD. CD25, CD80, CD95, HLA-DR were used to assess B-cell activation. Intracellular production of IL-10 and IL-6 were assessed by flow cytometry after TLR9 and CD40 stimulation. IL-6, IL-10, Ki67, Bcl2 mRNA were quantified in B-cells. Cytokine production was also assessed in sera and supernatants of B-cell culture, using a multiplex approach. Signaling pathways were studied through phosphorylation of mTOR, ERK, STAT3, STAT5 using a flow cytometry approach.We found that SSc patients exhibited an altered peripheral blood B-cell subset distribution, with decreased memory B-cells but increased proportion of naive and CD21^LoCD38^Lo B-cell subsets. We observed an increased expression of activation markers (CD80, CD95, HLA-DR) on some B-cell subsets, mainly the memory B-cells. Secretion of IL-6, BAFF and CXCL13 were increased in SSc sera. There was no correlation between the peripheral blood B-cell subsets and the serum concentrations of these cytokines. After stimulation, we observed a lower proportion of IL-10 and IL-6 producing B–cells in SSc. Finally, we observed a significant decrease of mTOR phosphorylation in SSc patient B-cells.In conclusion, we observed an altered B-cell homeostasis in SSc patients compared to HC. Memory B-cells were both decreased and activated in patients. IL-10 producing B-cells were decreased in SSc. This decrease was associated with an alteration of mTOR phosphorylation in B-cells. Conversely, there was no correlation between serum cytokine profile and B-cell homeostasis alterations.     

Consequences of viral infections for lymphocyte compartmentalization and homeostasis

The immune system has evolved to deal with pathogens. Analysing what happens during the course of infectious processes provides insights into the limits of lymphocyte homeostasis. Virus infections greatly alter normal T- and B-cell prevalence and localization patterns. Any mechanism that ‘counts’ T cells and B cells seems to be disrupted, at least while antigen persists. There is no simple ‘dumping’ process that controls numbers in the blood. Though the cell-surface ‘language’ that determines lymphocyte trafficking patterns must be central to modulating the consequences of infectious diseases, it is far from clear how such interactions maintain the system in reasonable balance.     

Cellular cytotoxicity generated in a canine mixed kidney lymphocyte cell culture

Canine cultured kidney epithelial cells were stimulatory to allogeneic lymphocytes in mixed kidney cell - lymphocyte cultures (MKLC). Generation of cytotoxic cells, cytotoxic for both kidney cells and PHA stimulated lymphoblasts of the stimulator have been observed. Lymphocyte stimulation and generation of CTL occurred in the MKLC at lower stimulator: responder cell ratios as in the mixed lymphocyte culture (MLC).     

IgG-mediated cytotoxicity to myenteric plexus cultures in patients with paraneoplastic neurological syndromes

Autoantibodies against neuronal and tumour proteins have been described in many paraneoplastic neurological syndromes (PNS), but it is not clear whether these antibodies are pathogenic or simply a useful diagnostic tool. We took seven sera that were positive on routine screening for antineuronal antibodies and the IgG fractions. As controls we used sera from health blood-donors, other neurological autoimmune diseases and patients with SCLC without PNS. We tested them on dissociated rat myenteric plexus cultures for cytotoxic effects. After incubation for 24 h, cytotoxicity was determined by a double fluorescence test (calcein green for living cells and ethidium homodimer-1 for dead cells). We found an increased cell death rate in cultures incubated with the PNS sera, compared with all controls (P< 0.05). Isolated IgG fractions were also cytotoxic whereas the IgG-free serum fraction did not show any significant increase in cytotoxicity. After incubation with PNS IgG, FACS analysis revealed an increased cytotoxicity rate only of the neurones, but not the glial cells. Our results indicate that in PNS a complement-independent, antibody-mediated cytotoxicity against neurones may contribute to the pathogenesis of these syndromes.     

Antibody-dependent and phytohaemagglutinin-induced lymphocyte cytotoxicity in systemic sclerosis.

Cell-mediated cytotoxicity was examined in thirty-seven patients with systemic sclerosis using both whole blood and purified peripheral blood mononuclear cells (PBM) to measure antibody-dependent (ADCC) and phytohaemagglutinin (PHA) induced lymphocyte cytotoxicity to 51Cr-labelled Chang liver cells. In twenty-three mildly affected patients, ADCC and PHA-induced cytotoxicity did not differ from that found in control populations. By contrast, fourteen patients severely affected by extensive visceral disease showed reductions in both ADCC and PHA-induced cytotoxicity which were more marked in whole blood assays (P less than 0.001) than in those performed with PBM (P less than 0.05). The addition of patient's sera to control cytotoxicity assays suggested that blocking or suppressive serum factors could only account for some of the disproportionate reduction in whole blood cytotoxicity which, in the main, must be due to a lack of circulating effector cells. These results are in agreement with previous findings of reduced numbers of circulating thymus-dependent lymphocytes in patients with severe disease, a defect of cell-mediated immunity that may result from the chronic antigenic stimulation of an autoimmune disease process.     

Angiopoietins: a link between angiogenesis and inflammation

The angiopoietin (Ang)-Tie ligand-receptor system has a key regulatory role in regulating vascular integrity and quiescence. Besides its role in angiogenesis, it is an important regulator in numerous diseases including inflammation. Ang-1-mediated Tie2 activation is required to maintain the quiescent resting state of the endothelium. Agonistic Ang-1 functions are antagonized by Ang-2, which is believed to inhibit Ang-1-Tie2 signaling. Ang-2 destabilizes the quiescent endothelium and primes it to respond to exogenous stimuli, thereby facilitating the activities of inflammatory (tumor necrosis factor and interleukin-1) and angiogenic (vascular endothelial growth factor) cytokines. Intriguingly, Ang-2 is expressed weakly by the resting endothelium but becomes strongly upregulated following endothelial activation. Moreover, endothelial cells store Ang-2 in Weibel-Palade bodies from where it can be made available quickly following stimulation, suggesting a role of Ang-2 in controlling rapid vascular adaptive processes. This suggests that Ang-2 is the dynamic regulator of the Ang-Tie2 axis, thereby functioning as a built-in switch controlling the transition of the resting quiescent endothelium towards the activated responsive endothelium.     

A link between fertility and K+ homeostasis: role of the renal H,K-ATPase type 2

Renal K⁺ retention is activated during pregnancy through a mechanism unknown to date. Here, we showed that the renal stimulation of H,K-ATPase type 2 (HKA2), whose expression was recently identified to be progesterone-dependent, is part of the mechanism favoring K⁺ accumulation during gestation. Moreover, investigation of the gestational phenotype of HKA2-null mice compared to their wild-type (WT) littermate revealed a decrease in fertility (gestation was successful in 33 % of HKA2-null mice vs. 83 % of WT mice) and in litter size (6.5?±?0.6 and 7.8?±?0.4 fetuses per litter, respectively). We also observed that urinary K⁺ excretion decreased by 20 % and plasma K⁺ concentration rose slightly (11 %) in WT mice during gestation (relative to basal conditions). In contrast, the renal excretion of K⁺ and plasma K⁺ levels in HKA2-null mice remained constant during gestation, whereas fecal K⁺ excretion increased. As a consequence, HKA2-null mice did not accumulate K⁺ in their extracellular compartment as efficiently as WT mice did. Finally, the link between inefficient K⁺ balance adaptations and gestational complications was established when we observed that these complications could be reversed with an increased K⁺ uptake. Altogether, these results define a novel physiological role for the HKA2 transporter and uncover a link between K⁺ metabolism and fertility.     

Lactoferrin-inducible monocyte cytotoxicity for K562 cells and decay of natural killer lymphocyte cytotoxicity.

Monocyte-enriched and lymphocyte-enriched fractions of peripheral blood from three healthy volunteers were obtained by percoll density gradient centrifugation. The cytotoxic activity of each fraction against 51Cr-labelled K562 cells was quantified in a 2-h assay using freshly isolated cells of each fraction and cells of each fraction which had been incubated with and without lactoferrin in complete medium for 18 h before performing the assay. We have thereby shown that cytotoxicity was not demonstrable in the lymphocyte fraction (containing 7.3 +/- 2% large granular lymphocytes) after 18 h in medium, whereas the cytotoxicity of the monocyte fraction (containing 3 +/- 0.4% large granular lymphocytes) was still significantly increased (P less than or equal to 0.01) and that lactoferrin had no effect on lymphocyte fraction cytotoxicity while producing an 11-fold increase in the cytotoxicity of the monocyte fraction. It is therefore possible to perform a relatively simple test of monocyte cytotoxicity using lactoferrin as a stimulant in a 2-h 51Cr-labelled K562 assay system by allowing 18 h to elapse for lymphocyte natural killer cytotoxicity to decay.     

Inhibition of human monocyte-macrophage and lymphocyte cytotoxicity to herpes simplex-infected cells by glucan.

The effect of short term in vitro incubation of glucan--a reticuloendothelial system stimulator--on subsequent cytotoxicity of human monocyte-macrophages (MP) and lymphocytes (L) to Herpes simplex virus-infected cells in a 51Cr-release assay was analyzed. Particulate, cell-associated glucan irreversibly inhibited MP antibody-dependent cellular cytotoxicity (ADCC). In contrast, the inhibition of L-ADCC and L-natural killer cytotoxicity could be reversed by dissociation of glucan and cells utilizing serum gradient centrifugation, a process which did not remove the glucan. These experiments reveal further basic differences between MP and L-ADCC using the reagent glucan.