Journal of Leukocyte Biology Cell Development, Growth, Differentiation, and Function

[Cell Development, Growth, Differentiation, and Function] The frequency of regulatory CD3+CD8+CD28-CD25+ T lymphocytes in human peripheral blood increases with age

Simone, R., Zicca, A., Saverino, D. — 2008-11-19

Aging is commonly associated with immune deficiency and dysregulation. The aging of the immune system involves a progressive reduction in naïve T cell output associated with thymic involution and peripheral expansion of oligoclonal memory T cells. We have investigated frequency, phenotype, and function of CD3⁺CD8⁺CD28^–CD25⁺ T cells in healthy volunteers over a wide age range. We demonstrate that the frequency of CD3⁺CD8⁺CD28^–CD25⁺ T cells in healthy volunteers increases with age. Peripheral CD3⁺CD8⁺CD28^–CD25⁺ T cells share phenotypic and functional features with CD3⁺CD4⁺CD25⁺ regulatory T cells (Tregs): In particular, they strongly express CTLA-4 and forkhead box P3. We observed that in vitro, functional titration assays of CD3⁺CD8⁺CD28^–CD25⁺ T cells show equivalent regulatory function in young and elderly donors, with suppression of proliferation and cytokine production in response to polyclonal T cell stimulation. Finally, CD3⁺CD8⁺CD28^–CD25⁺ T cells seem to specifically express the CD122 receptor. Altogether, these observations demonstrate an increase in peripheral blood CD8⁺ Tregs associated with aging.

[Cell Development, Growth, Differentiation, and Function] Bacterial infection alters the kinetics and function of iNKT cell responses

Choi, H.-J., Xu, H., Geng, Y., Colmone, A., Cho, H., Wang, C.-R. — 2008-11-19

CD1d-restricted V14 invariant NKT cells (iNKT) are innate-like, immunoregulatory lymphocytes that play critical roles in autoimmunity, tumor surveillance, and infectious disease. Although iNKT cells are activated during microbial infection, the impacts of infection on the function of iNKT cells have not been fully characterized. Using a Listeria monocytogenes (LM) infection model, we found that iNKT cells failed to expand after infection, resulting in prolonged loss in the spleen, in contrast to the typical expansion and contraction of conventional T cells. iNKT cells from LM-infected mice responded more rapidly to secondary LM infection; however, they became functionally hyporesponsive to antigenic challenge for at least 1 month. This infection-induced hyporesponsiveness was also induced by Mycobacteria infection and was more profound in LM-infected, thymectomized mice, suggesting that infection-primed iNKT cells might have altered functionality. Interestingly, activation with -galactosylceramide-loaded dendritic cells was able to overcome infection-induced hyporesponsiveness of iNKT cells, suggesting a role for extrinsic factors in this functional deficit. Taken together, these findings suggest that infection affects iNKT cell responses quantitatively and qualitatively. As humans are under constant microbial insult, predictions of iNKT cell function based on naïve animal models may not accurately reflect iNKT cell behavior in a clinical setting.

[Cell Development, Growth, Differentiation, and Function] Human dendritic cells differentiated in hypoxia down-modulate antigen uptake and change their chemokine expression profile

2008-11-19

Dendritic cells (DCs) are the most potent antigen-presenting cells and fine-tune the immune response. We have investigated hypoxia’s effects on the differentiation and maturation of DCs from human monocytes in vitro, and have shown that it affects DC functions. Hypoxic immature DCs (H-iDCs) significantly fail to capture antigens through down-modulation of the RhoA/Ezrin-Radixin-Moesin pathway and the expression of CD206. Moreover, H-iDCs released higher levels of CXCL1, VEGF, CCL20, CXCL8, and CXCL10 but decreased levels of CCL2 and CCL18, which predict a different ability to recruit neutrophils rather than monocytes and create a proinflammatory and proangiogenic environment. By contrast, hypoxia has no effect on DC maturation. Hypoxic mature DCs display a mature phenotype and activate both allogeneic and specific T cells like normoxic mDCs. This study provides the first demonstration that hypoxia inhibits antigen uptake by DCs and profoundly changes the DC chemokine expression profile and may have a critical role in DC differentiation, adaptation, and activation in inflamed tissues.

[Cell Development, Growth, Differentiation, and Function] Regulation of WAVE1 expression in macrophages at multiple levels

Dinh, H., Scholz, G. M., Hamilton, J. A. — 2008-11-19

M-CSF (or CSF-1) controls macrophage lineage development and function. A CSF-1-dependent culture system was established, which monitored the differentiation of CSF-1-responsive macrophage populations over time and upon adherence. Wiskott-Aldrich syndrome protein verprolin homologous (WAVE) proteins are involved in actin reorganization, a process critical to many cell functions. WAVE2 but not WAVE1 has been considered significant for macrophage function. Using the CSF-1-dependent differentiation system, we were able to demonstrate the contrasting regulation of the expression of WAVE1 and WAVE2; the levels of the latter rose over time and as the macrophage population became adherent, although those of the former increased over time but were down-regulated upon adherence. Evidence was obtained that WAVE1 was also cleaved to a novel, 60-kDa fragment by macrophage adherence and by another pathway involving calpain-mediated proteolysis. Mutagenesis studies indicated that cleavage of WAVE1 by calpain results in the removal of the verprolin-homology, cofilin-like, and acidic domain and thus, the loss of WAVE1 activity. We suggest that WAVE1 is also important for macrophage biology and that it could have separate functions to those of WAVE2.

[Cell Development, Growth, Differentiation, and Function] Functional expression of IgA receptor Fc{alpha}RI on human platelets

Qian, K., Xie, F., Gibson, A. W., Edberg, J. C., Kimberly, R. P., Wu, J. — 2008-11-19

FcRI (CD89) is a human IgA FcR expressed on cells of myeloid lineage such as neutrophils, monocytes, tissue macrophages, eosinophils, and subpopulations of dendritic cells. FcRI mediates cell activation through Src family kinases and downstream tyrosine-based phosphorylation pathways. However, the role of IgA and the expression and role of its cognate receptor FcRI (CD89) in platelet activation are undefined. In the current study, we demonstrate that human platelets express FcRI mRNAs and proteins. Furthermore, we show that the platelet FcRI is associated with the FcR -chain, and cross-linking of FcRI leads to Syk phosphorylation. Clustering of FcRI induces pre-mRNA splicing and protein production of tissue factor and IL-1β, suggesting novel roles for human platelet FcRI and serum IgA in thrombosis and inflammation.

[Cell Development, Growth, Differentiation, and Function] CD5-low expression lymphocytes in canine peripheral blood show characteristics of natural killer cells

2008-11-19

NK cell markers and receptors have been discovered in many mammalian species, such as humans, mice, rats, pigs, and cows. However, there is still a lack of information concerning NK cell markers or receptors in canines. We have discovered that canine CD5-low density (CD5^lo) cells in PBL are closely associated with NK cell characteristics. CD5^lo cells comprised 14.9 ± 6.68% of the total PBL. A high proportion of the CD5^lo cell population expressed CD3 (96.6%), CD8 (77.7%), CD8β (53%), /β TCR (83%), and CD11/18 (80%), but the expression of / TCR (6.5%), CD4 (10.6%), and CD21 (2.4%) was low. CD5^lo cells were larger than CD5-high density (CD5^hi) cells. Light and electron microscopy revealed numerous large cytoplasmic granules in CD5^lo cells, especially after IL-2 stimulation, which was in contrast to CD5^hi, in which intracytoplasmic granules were not frequently seen. After IL-2 stimulation, CD5^lo cells had significantly stronger NK cytotoxicity than CD5^hi cells. CD5^lo cells had much higher mRNA levels for NKG2D, CD16, CD94, CD160, perforin, and granzyme than CD5^hi. Following IL-2 stimulation, CD5^lo cells had significantly higher mRNA levels of NKp30, NKp44, CD16, and CD94 than CD5^hi cells. In addition, IL-2-stimulated, CD5^lo-depleted PBL showed a loss of NK cytotoxicity. CD5^lo cells also showed significantly lower antigen-specific cytotoxic T cell activity as compared with CD5^hi cells. Taken together, the CD5^lo subset in canine PBL is closely related to canine NK cells, and CD5^lo can be used as a phenotypic marker for an IL-2-dependent canine NK cell enrichment.

[Cell Development, Growth, Differentiation, and Function] A novel subset of NK cells expressing high levels of inhibitory Fc{gamma}RIIB modulating antibody-dependent function

2008-11-19

NK cells can kill antibody-coated target cells following engagement of FcRIIIA, the major activating FcR expressed by these cells. The presence of FcRIIC (CD32C) has also been reported, but its contribution to the FcR-dependent effector functions of NK cells remains debated. We demonstrate here that inhibitory FcRIIB is also expressed by a small subset of CD56⁺/NKp46⁺ NK cells and can efficiently down-modulate their FcR-dependent effector function. Immunofluorescence analyses of NK cells from 52 healthy donors showed the presence of CD56^bright/FcRII^– (5.2%±3.4), CD56^dim/FcRII^lo/- (94.1%±3.4), and CD56^dim/FcRII^bright (0.64%±0.72) cells. QRT-PCR and protein analyses performed on isolated FcRII^bright NK cells indicated that FcRIIB is strongly expressed by these cells but not by FcRII^lo/- cells. In addition, FcRII^bright cells showed a weaker antibody-dependent degranulation when incubated with IgG-coated target cells compared with FcRII^lo/- NK cells, although a strong FcRIIIA expression was detected in both cells. Furthermore, the addition of anti-FcRII Fab paralleled a higher degranulation of FcRII^bright NK cells, indicating a direct role for FcRIIB in this down-modulating effect. Thus, it is proposed that FcRIIB^bright NK cells represent a new NK cell compartment able to down-modulate NK cell functions triggered by the engagement of activating FcR.

[Cell Development, Growth, Differentiation, and Function] Recent thymic origin, differentiation, and turnover of regulatory T cells

Mabarrack, N. H. E., Turner, N. L., Mayrhofer, G. — 2008-10-14

Regulatory CD4⁺ T cells (T_reg) are essential to maintain self-tolerance. Release of natural T_reg from the thymus is believed to commence soon after birth, but it is unclear how many are produced by "conversion" in the periphery, whether numbers are maintained after puberty by general homeostatic mechanisms that regulate lymphocyte numbers, or whether significant numbers are produced by the involuted thymus. To address the origin of T_reg in normal adult rats, we focused on recent thymus emigrants (RTE). Approximately 30% of CD4⁺CD25⁺forkhead box p3 (Foxp3)⁺ T_reg expressed markers associated with RTE. Following thymectomy, numbers of cells expressing these markers fell by 80% within 30 days. Furthermore, although only ~5% of CD4⁺ single-positive thymocytes expressed Foxp3 within 24 h after intrathymic injection of FITC, more than 30% of the labeled CD4⁺ RTE were Foxp3⁺, suggesting that some RTE may acquire Foxp3 in the periphery. Thus, some RTE may acquire Foxp3 rapidly after emigration from the thymus. T_reg are dividing rapidly with apparent half-lives of ~18 days and ~7 days for the CD4⁺CD25⁺Foxp3⁺ and CD4⁺CD25^–Foxp3⁺ subsets, respectively. The apparently slower turnover of CD4⁺CD25⁺Foxp3⁺ cells is a result of CD4⁺CD25⁺Foxp3⁺ -> CD4⁺CD25^–Foxp3⁺ conversion, with no loss of regulatory function. Taken together, the data suggest that T_reg in adults are relatively short-lived and that their numbers are maintained by rapid cell division and continuous replenishment from the thymus.

[Cell Development, Growth, Differentiation, and Function] Trogocytosis and killing of IL-4-polarized monocytes by autologous NK cells

Poupot, M., Fournie, J.-J., Poupot, R. — 2008-10-14

Cross-regulations between innate immune cells have been given more and more emphasis. Here, we address the question of bidirectional interactions between activated monocytes and autologous NK cells. Classically activated monocytes (class-monocytes), obtained by priming with IFN-, drive an inflammatory immune response. On the contrary, alternatively activated monocytes (alt-monocytes), obtained by stimulation with IL-4 or IL-13, engage an anti-inflammatory immune response. We show that alt-monocytes inhibit proliferation and production of IFN- by autologous, IL-2-activated NK cells, whereas class-monocytes do not inhibit these NK cell functions. Reciprocally, IL-2-activated NK cells interact and undertake intensive synaptic transfer with alt-monocytes, whereas interactions with class-monocytes are weaker. This strong trogocytosis correlates with an efficient killing of alt-monocytes, mediated by natural cytotoxicity receptors and a lowered killing of class-monocytes. These results suggest that interactions between NK cells and autologous-activated monocytes modulate inflammatory responses. This might be extended further in the elimination of tumor-associated macrophages, which actively promote solid tumor progression and metastasis.

[Cell Development, Growth, Differentiation, and Function] Maturation of dendritic cells depends on proteolytic cleavage by cathepsin X

Obermajer, N., Svajger, U., Bogyo, M., Jeras, M., Kos, J. — 2008-10-14

The maturation status of dendritic cells (DCs) is crucial for effective antigen presentation and initiation of the primary immune response. Maturation stimuli cause the adhesion of immature DCs to the extracellular matrix, which is accompanied by recruitment of the CD11b/CD18 [macrophage antigen-1 (Mac-1)] integrin receptor, cytoskeleton reorganization, and podosome formation. Cathepsin X, a cysteine protease expressed in DCs and other APCs, is involved in Mac-1 activation. We have shown that during maturation, cathepsin X translocates to the plasma membrane of maturing DCs, enabling Mac-1 activation and consequently, cell adhesion. In mature DCs, cathepsin X redistributes from the membrane to the perinuclear region, which coincides with the de-adhesion of DCs, formation of cell clusters, and acquisition of the mature phenotype. Inhibition of cathepsin X activity during DC differentiation and maturation resulted in an altered phenotype and function of mature DCs. It reduced surface expression of costimulatory molecules, increased expression of inhibitory Ig-like transcripts 3 and 4 (ILT3 and ILT4), almost completely abolished cytokine production, diminished migration, and reduced the capacity of DCs to stimulate T lymphocytes. These results stress the importance of cathepsin X in regulating DC adhesion, a crucial event for their maturation and T cell activation.

[Cell Development, Growth, Differentiation, and Function] IL-15-induced CD8+CD122+ T cells increase antibacterial and anti-tumor immune responses: implications for immune function in aged mice

Motegi, A., Kinoshita, M., Inatsu, A., Habu, Y., Saitoh, D., Seki, S. — 2008-09-30

We previously proposed that mouse CD8⁺CD122⁺ T cells and human CD57⁺ T cells, which increase with age and exhibit potent IFN- production, represent a double-edged sword as they play critical roles in host defense and the lethal IL-12/LPS-induced generalized Shwartzman reaction (GSR). However, our proposal was based solely on comparisons of young and old mice. In this study, we attempted to increase CD8⁺CD122⁺ T cells in young mice with exogenous IL-15 and confirm their countervailing functions in young mice. After young mice (6 weeks) were injected with IL-15, they showed significant increases in CD8⁺CD122⁺ T cells in the liver and spleen. Liver CD8⁺CD122⁺ T cells from IL-15-pretreated mice had a potent capacity to produce IFN- after IL-12 injection or Escherichia coli infection. IL-15-pretreated mice showed increased survival to E. coli infections and enhanced anti-tumor activities against liver metastatic EL4 cells, as well as an exacerbation of the GSR. Correspondingly, liver CD8⁺CD122⁺ T cells produced more perforin than CD8⁺CD122^– T cells in EL4-inoculated mice. Unexpectedly, comparable IL-15 treatment did not induce further increases in CD8⁺CD122⁺ T cells in aged mice and did not enhance their defenses against bacterial infection or tumor growth. Interestingly, however, nontreated, aged mice (50 weeks) showed twofold higher IL-15 levels (but not TNF or IFN-) in liver homogenates compared with young mice. Our results further support that CD8⁺CD122⁺ T cells, which are increased physiologically or therapeutically by IL-15, are involved in antibacterial immunity, anti-tumor immunity, and the GSR.

[Cell Development, Growth, Differentiation, and Function] Induction of human CD4+ regulatory T cells by mycophenolic acid-treated dendritic cells

2008-09-30

Depending on their degree of maturation, costimulatory molecule expression, and cytokine secretion, dendritic cells (DC) can induce immunity or tolerance. DC treated with mycophenolic acid during their maturation (MPA-DC) have a regulatory phenotype and may therefore provide a new approach to induce allograft tolerance. Purified CD4⁺ T cells stimulated in a human in vitro model of mixed culture by allogeneic MPA-DC displayed much weaker proliferation than T cells activated by mature DC and were anergic. This hyporesponsiveness was alloantigen-specific. Interestingly, T cells stimulated by MPA-DC during long-term coculture in four 7-day cycles displayed potent, suppressive activity, as revealed by marked inhibition of the proliferation of naive and preactivated control T cells. These regulatory T cells (Tregs) appeared to have antigen specificity and were contact-dependent. Tregs induced by MPA-DC were CD25⁺glucocorticoid-induced TNFR⁺CTLA-4⁺CD95⁺, secreted IL-5 and large amounts of IL-10 and TGF-β, and displayed enhanced forkhead box p3 expression. These results obtained in vitro demonstrate that human MPA-DC can induce allospecific Tregs that may be exploited in cell therapy to induce allograft tolerance.

[Cell Development, Growth, Differentiation, and Function] CD56bright cells increase expression of {alpha}4 integrin at ovulation in fertile cycles

Peralta, C. G., Han, V. K., Horrocks, J., Croy, B. A., van den Heuvel, M. J. — 2008-09-30

Leukocyte content of human endometrium changes rapidly after ovulation, particularly as a result of gains in CD56^bright uterine NK (uNK) cells. We have proposed that uNK precursor cells are found within the blood CD56^bright pool and are recruited to decidualizing endometrium through functional changes in their adhesion molecules and chemokine receptors. This study sought to quantify alterations in adhesion molecules, cytokines, chemokines, and receptors induced in circulating CD56⁺ cells of fertile and infertile women by ovulation. Blood was drawn from 12 fertile volunteers and six female-infertility patients at Menstrual Cycle Day (d) 5 and on the day following the preovulatory surge of luteinizing hormone (LH). CD56^bright, CD56^dim, and CD56⁺CD3⁺ cell subsets were isolated and evaluated by flow cytometry, quantitative PCR, or Western blotting. In CD56^bright cells from fertile but not infertile women, ₄ integrin increased between d5 and the preovulatory LH surge. CD56^dim and NKT cells did not show a change in ₄ integrin but differed significantly between fertile and infertile donors, and infertile donors had reduced homing molecule expression in CD56^dim and NKT cells, and at ovulation, their NKT cells showed elevated cytokine production. None of the circulating CD56⁺ cell subsets had transcripts for receptors for estrogen, progesterone, LH, or prolactin. Thus, immunological events associated with the LH surge induce alterations in all subsets of CD56⁺ cells, and the unique induction of ₄ integrin in CD56^bright cells of fertile women constitutes a potential method to promote uterine homing.

[Cell Development, Growth, Differentiation, and Function] Expression of sialyltransferase activity on intact human neutrophils

2008-09-30

Endogenous polymorphonuclear leukocyte (PMN)-associated sialidase activity enhances PMN adhesion to and migration across the endothelium through the removal of sialylated cell-surface residues. We tested the hypothesis that PMNs also express sialyltransferase (ST) activity that restores sialyl residues to the PMN surface. We developed a highly sensitive fluorometric assay to demonstrate that intact human PMNs can mediate and accept sialyl residue transfer. This ST activity is inhibited by a ST inhibitor, CMP, which also inhibits the transendothelial migration of PMNs in response to IL-8 in vitro and in vivo. We conclude that intact PMNs express sialidase and ST activities that permit rapid modulation of their surface sialylation and their ability to adhere to and migrate across the endothelium.

[Cell Development, Growth, Differentiation, and Function] Differential turnover rates of monocyte-derived cells in varied ocular tissue microenvironments

Kezic, J., McMenamin, P. G. — 2008-08-18

Monocytes of bone marrow (BM) origin are circulating precursors that replenish dendritic cells and macrophage populations in peripheral tissues during homeostasis. The eye provides a unique range of varying tissue microenvironments in which to compare the different turnover rates of monocyte-derived cells. This was investigated in the present study using radiation chimeras, whereby BM from Cx3cr1^+/gfp mice was used to rescue myeloablated wild-type (WT) BALB/c mice (conventional chimeras). The use of Cx3cr1^+/gfp mice as BM donors allowed the clear visualization of newly recruited monocyte-derived cells. Following BM reconstitution, mice were killed at 2, 4, 6, and 8 weeks, and wholemount ocular tissues were processed for immunohistochemistry and confocal microscopy. "Reverse" chimeras (WT into Cx3cr1^+/gfp) were also created to act as a further method of cross-referencing cell turnover rates. In conventional chimeras, Cx3cr1^+/gfp cells began repopulating the uveal tract (iris, ciliary body, choroid) 2 weeks post-transplantation with close to complete replenishment by 8 weeks. By contrast, the earliest recruitment of Cx3cr1^+/gfp cells into the host retina occurred at 4 weeks. In reverse chimeras, a steady accumulation of host Cx3cr1^+/gfp macrophages in the subretinal space of Cx3cr1^+/gfp adult mice suggests that these cells arise from long-term resident microglia and not newly recruited WT donor cells. In summary, chimeric mouse models, in which lineage-specific cells carry a fluorescent reporter, have been used in the present study to visualize the turnover of monocyte-derived cells in different tissue compartments of the eye. These data provide valuable insights into differential monocyte turnover rates within a single complex organ.

[Cell Development, Growth, Differentiation, and Function] Sialyl lewisx antigen-expressing human CD4+ T and CD8+ T cells as initial immune responders in memory phenotype subsets

2008-08-18

Cytokine production by memory T cells in secondary immune responses has a critical role in host defenses. Previously, we had demonstrated that a unique antigen composed of sialyl lewis^x (sLe^x) was expressed on CD45RO⁺ memory-phenotype subsets of human T cells. Here, we found that the sLe^x antigen was up-regulated on CD45RA⁺ naïve human CD4⁺ T and CD8⁺ T cells by TCR stimulation. In addition, sLe^x antigen-expressing CD4⁺ T and CD8⁺ T cells in human PBMCs were activated immediately by cytokine stimulations composed of IL-2 plus IL-12 or IL-15 in an antigen-independent manner. Moreover, the sLe^x-positive human CD8⁺ T cells significantly enhanced reverse antibody-dependent cellular cytotoxicity compared with a sLe^x-negative population. These findings clearly indicate that sLe^x antigen-expressing memory phenotype CD4⁺ T and CD8⁺ T cells contribute to early-stage immunity by providing a source of IFN- and cytotoxicity, suggesting that they would be a key immunomodulator in host defenses.