Age-associated alterations in CXCL1 chemokine expression by murine B cells

BACKGROUND: The CXCL1 chemokines, macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (KC), have been shown to play a role in a number of pathophysiological disease states including endotoxin-induced inflammation and bacterial meningitis. While the expression of these chemokines has been identified in a variety of cell types in the mouse, little is known about their expression with murine B-lymphocytes. RESULTS: Here, we demonstrate that highly purified murine splenic B cells are capable of expressing both MIP-2 and KC protein and mRNA upon activation with lipopolysaccharide (LPS) but not in response to anti-μ and anti-CD40 in combination with interleukin-4 (IL-4) stimulation. Moreover, these chemokines are expressed at higher levels in B cells derived from young (4 m) compared to old (24–29 m) mice. Upon fractionation into distinct B-cell subsets, we found that the expression of MIP-2 and KC by aged follicular (FO) B cells is significantly decreased when compared to the same cells from younger mice, while only MIP-2 production was found to be diminished in aged marginal zone (MZ) B cells. Interestingly, MIP-2 and KC production by newly formed (NF) B cells did not significantly differ with age. Moreover, the potential relevance of these findings is supported by the poor ability of LPS-activated aged B cells to specifically mediate CXCL1-dependent leukocyte recruitment when compared to younger B cells. CONCLUSION: Overall, the decreased expression of CXCL1 chemokines by aged B cells in response to LPS may have potential implications on the secondary recruitment of leukocytes to sites of microbial infections and inflammation possibly contributing to the increased susceptibility of older subjects to pathogen challenge

Quantitative differences in lipid raft components between murine CD4(+ )and CD8(+ )T cells

BACKGROUND: Lipid rafts have been shown to play a role in T cell maturation, activation as well as in the formation of immunological synapses in CD4(+ )helper and CD8(+ )cytotoxic T cells. However, the differential expression of lipid raft components between CD4(+ )and CD8(+ )T cells is still poorly defined. To examine this question, we analyzed the expression of GM1 in T cells from young and aged mice as well as the expression of the glycosylphosphatidylinositol (GPI)-linked protein Thy-1 and cholesterol in murine CD4(+ )and CD8(+ )T cell subpopulations. RESULTS: We found that CD4(+)CD8(- )and CD8(+)CD4(- )thymocytes at different stages of maturation display distinct GM1 surface expression. This phenomenon did not change with progressive aging, as these findings were consistent over the lifespan of the mouse. In the periphery, CD8(+ )T cells express significantly higher levels of GM1 than CD4(+ )T cells. In addition, we observed that GM1 levels increase over aging on CD8(+ )T cells but not in CD4(+ )T cells. We also verified that naïve (CD44(lo)) and memory (CD44(hi)) CD8(+ )T cells as well as naïve and memory CD4(+ )T cells express similar levels of GM1 on their surface. Furthermore, we found that CD8(+ )T cells express higher levels of the GPI-anchored cell surface protein Thy-1 associated with lipid raft domains as compared to CD4(+ )T cells. Finally, we observed higher levels of total cellular cholesterol in CD8(+ )T cells than CD4(+ )T cells. CONCLUSION: These results demonstrate heterogeneity of lipid raft components between CD4(+ )and CD8(+ )T cells in young and aged mice. Such differences in lipid raft composition may contribute to the differential CD4 and CD8 molecule signaling pathways as well as possibly to the effector responses mediated by these T cell subsets following TCR activation

Lipid-laden multilocular cells in the aging thymus are phenotypically heterogeneous

Intrathymic lipid-laden multilocular cells (LLMC) are known to express pro-inflammatory factors that might regulate functional activity of the thymus. However, the phenotype of ageassociated intrathymic LLMC is still controversial. In this study, we evaluated LLMC density in the aging thymus and better characterized their distribution, ultrastructure and phenotype. Our results show an increased density of LLMC in the thymus from 03 to 24 months of age. Morphologically, intrathymic LLMC exhibit fibroblastoid fusiform, globular or stellate shapes and can be found in the subcapsular region as well as deeper in the parenchyma, including the perivascular area. Some parenchymal LLMC were like telocytes accumulating lipids. We identified lipid droplets with different electrondensities, lipofuscin granules and autolipophagosomelike structures, indicating heterogeneous lipid content in these cells. Autophagosome formation in intrathymic LLMC was confirmed by positive staining for beclin-1 and perilipin (PLIN), marker for lipid droplet-associated proteins.We also found LLMC in close apposition to thymic stromal cells, endothelial cells, mast cells and lymphocytes. Phenotypically, we identified intrathymic LLMC as preadipocytes (PLIN+PPARγ2+), brown adipocytes (PLIN+UCP1+), macrophages (PLIN+Iba-1+) or pericytes (PLIN+NG2+) but not epithelial cells (PLIN- panCK+). These data indicate that intrathymic LLMC are already present in the young thymus and their density significantly increases with age. We also suggest that LLMC, which are morphologically distinct, establish direct contact with lymphocytes and interact with stromal cells. Finally, we evidence that intrathymic LLMC correspond to not only one but to distinct cell types accumulating lipids

Fat-Storing Multilocular Cells Expressing CCR5 Increase in the Thymus with Advancing Age: Potential Role for CCR5 Ligands on the Differentiation and Migration of Preadipocytes

<p>Age-associated thymic involution is characterized by decreased thymopoiesis, adipocyte deposition and changes in the expression of various thymic microenvironmental factors. In this work, we characterized the distribution of fat-storing cells within the aging thymus. We found an increase of unilocular adipocytes, ERTR7⁺ and CCR5⁺fat-storing multilocular cells in the thymic septa and parenchymal regions, thus suggesting that mesenchymal cells could be immigrating and differentiating in the aging thymus. We verified that the expression of CCR5 and its ligands, CCL3, CCL4 and CCL5, were increased in the thymus with age. Hypothesizing that the increased expression of chemokines and the CCR5 receptor may play a role in adipocyte recruitment and/or differentiation within the aging thymus, we examined the potential role for CCR5 signaling on adipocyte physiology using 3T3-L1 pre-adipocyte cell line. Increased expression of the adipocyte differentiation markers, PPAR&#947;2 and aP2 in 3T3-L1 cells was observed under treatment with CCR5 ligands. Moreover, 3T3-L1 cells demonstrated an ability to migrate<i> in vitro</i> in response to CCR5 ligands. We believe that the increased presence of fat-storing cells expressing CCR5 within the aging thymus strongly suggests that these cells may be an active component of the thymic stromal cell compartment in the physiology of thymic aging. Moreover, we found that adipocyte differentiation appear to be influenced by the proinflammatory chemokines, CCL3, CCL4 and CCL5.</p

Distribution, morphology and density analysis of LLMC in the aging thymus.

<p>Semi-thin thymic section of middle-aged mouse shows LLMC with brownish yellow color due to fixation with osmium-tetroxide. <b>A:</b> intrathymic LLMC presenting fusiform (black arrowhead), globular (yellow arrowhead) or stellate (white arrowhead) morphologies in the thymic parenchyma. <b>B:</b> LLMC adjacent to the thymic septa with fusiform (black arrowheads) and globular (yellow arrowhead) morphologies. <b>C:</b> Fusiform or Stellate pericyte-like LLMC (red arrowhead) visualized around vessels in the thymic parenchyma, adjacent to endothelial cells. <b>D</b>: Schematic representation showing distribution and morphologies of LLMC in the aged thymus. <b>E, F</b> and <b>G</b>: Oil Red O lipid staining of thymic cryosections from mice with 3, 12 and 24 months of age, respectively. ad: adipocyte; Ca: capsule. Scale bars in A: 20μm; B and C: 25μm; D, E, F: 30μm; <b>H</b>: Distribution and density of LLMC in the whole area of 5μm thymic tissue sections from mice with 3, 12 or 24 months of age (n = 4). C/S represents SC-LLMC adjacent to capsule or septa; V represents PV-Perivascular LLMC; and P represents P-LLMC, deeper in the parenchyma. <b>I</b>: Total lymphocyte number in the thymus of mice with 3, 12 or 24 months of age (n = 4, per each age group). Results are shown as mean ± SEM. *p<0.05; **p<0.005; ***p<0.0005.</p

Intrathymic LLMC present lipid droplets and vesicles of different electrondensities and autolipophagossome-like structures.

<p><b>A:</b> Upper panel shows intrathymic LLMC in middle-aged mouse. Semi-thin section fixed with osmium tetroxide. LLMC stained in brownish yellow. Lower panel shows TEM higher magnification of LLMC (false colored yellow) seen in the upper panel containing vesicles of different electrondensities. <b>B:</b> Left panel shows TEM photomicrograph of cytoplasmic lysosomes (indicated by black arrowheads) with lipofuscin and mixed content next to the LLMC nucleus (indicated by a black asterisk). Selected area with autolipophagosome-like multimembrane structure inside a lipid droplet is shown in higher magnification in the inset on the bottom left corner. Middle panel shows vesicles and lipid droplets of LLMC presenting electron-dense membranes typical of autolipophagosome formation. Small panels correspond to higher magnification of selected areas in the middle panel. White arrowheads indicate double membranes of autophagossomes with mixed content in lipid droplets. Right panel shows cytoplasmic lipofuscin granules and distinct electron-dense lipid droplets surrounded by a multi-laminar structure (indicated by a black arrowhead) in a LLMC. <b>C:</b> intrathymic LLMC (white arrow) positively labeled for Beclin-1 (green) and PLIN (red) by immunofluorescence. Cell nuclei labeled with Hoechst (blue). ld: lipid droplets. Scale bars in A, upper panel: 15μm, lower panel: 4μm; B left panel: 1μm, inset: 0.5μm; in B middle panel: 0.5μm; in B small panels: 0.1μm; in B right panel: 0.2μm; C: 15 μm.</p

Intrathymic LLMC possibly interact with lymphocytes, epithelial and endothelial cells.

<p><b>A:</b> Representative photomicrograph of fusiform SC-LLMC (black arrowhead); and globular (yellow arrowhead) or stellate (white arrowhead) P-LLMC in middle-aged mouse thymus section. Tissue fixation with osmium tetroxide reveals lipid-droplets in brownish yellow color. <b>B:</b> TEM photomicrographs show P-LLMC (false colored yellow), adjacent to subcapsular epithelial cells (SCE) (false colored blue), possibly interacting with lymphocytes in the thymic parenchyma. Selected traced area show SCE and LLMC in the limitation between parenchyma and capsule region (Ca); <b>C:</b> Higher magnification of the selected area in panel B shows a LLMC prolongation (false colored yellow) in the basal membrane between the two layers of SCE (false colored blue), in the limit of the thymic parenchyma. <b>D</b>: Semi-thin thymus tissue sections fixed with osmium tetroxide and counterstained with toluidine blue reveal globular P-LLMC (black asterisk) surrounding four lymphocytes. <b>E:</b> TEM photomicrograph showing a lymphocyte surrounded by the P-LLMC in Panel D. Selected traced area shows limiting areas between lymphocytes and LLMC (black asterisk in the nucleus); <b>F</b>: Higher magnification of the selected area in panel E shows the nucleus (white asterisk) and plasma membrane of a lymphocyte (black arrowheads) surrounded by the LLMC with a more electron-lucent cytoplasm. <b>G:</b> A LLMC (false colored yellow) embraces a lymphocyte with a telopode-like prolongation and possibly establishes membrane contact with another lymphocyte in the old thymus. A plasma cell (PL) is located adjacent to an intrathymic LLMC. <b>H:</b> Higher magnification of selected area in G shows possible membrane contact between LLMC and lymphocyte. Black arrowhead indicates close apposition between cell membranes; <b>I:</b> A P-LLMC (false colored yellow) possibly establishing contact with a lymphocyte. Inset on the bottom left corner demonstrates higher magnification of selected area showing close apposition between the cell membranes of LLMC and lymphocyte; <b>J:</b> P-LLMC (false colored yellow) showing a telopode-like prolongation embracing thymic lymphocytes. Telocyte podom-like structure is indicated (black arrowhead). <b>K:</b> TEM photomicrograph showing P-LLMC (false colored yellow) surrounding lymphocytes close to thymic epithelial cell (false colored blue) around the perivascular space (PVS); <b>L:</b> TEM photomicrograph showing LLMC (false colored yellow) in the PVS surrounded by epithelial cells (false colored blue). BV, blood vessel. Ca: capsule; PL: plasma cell; PVS: Perivascular space. Scale bars in A: 20 μm; B, G, J, L: 5 μm; C, E, K: 2 μm; D: 15 μm; F,H: 0.5 μm; I: 1μm and inset in I: 0.2 μm.</p

Pericyte-like LLMC possibly interacting with lymphocytes and endothelial cells in the aging thymus.

<p><b>A:</b> Semi-thin thymic section of middle-aged mice fixed with osmium tetroxide and stained with toluidine blue shows pericyte (green arrowhead) and pericyte-like LLMC (red arrowhead) possibly interacting with endothelial cells. <b>B:</b> TEM photomicrograph shows pericyte-like LLMC (false colored yellow) surrounding an endothelial cell and possibly interacting with various lymphocytes. White asterisk indicates elongated nucleus of a migrating lymphocyte. <b>C-E:</b> Higher magnifications of regions seen in B. Black arrowheads indicate close apposition between membranes of LLMC and thymic lymphocytes. In E, LLMC and endothelial cell share basal membrane. Asterisks indicate area of extracellular matrix that forms basal membrane <b>F:</b> PV-LLMC (false colored yellow) surrounding an endothelial cell and possibly interacting with lymphocytes in an old thymus. <b>G:</b> Higher magnification of selected area in F. Black arrowhead shows possible membrane contact between LLMC and lymphocyte. BV: blood vessel; P: pericyte. Scale bars in A: 15μm; B: 5μm; C, D: 1μm; and E: 2μm; F: 2μm and G: 400nm.</p