Increased lymphocytic aminopeptidase N/CD13 promoter activity after cell-cell contact

Aminopeptidase N (APN)/CD13 is a transmembrane ectoenzyme expressed on a wide variety of cells. With respect to haematopoietic cells, APN/CD13 has been considered specific for the myeloid lineage, because granulocytes and monocytes/macrophages, but not lymphocytes of peripheral blood, show a surface expression of CD13 antigen. However, we could recently show that cell-cell contact of lymphocytes with endothelial cells, monocytes, and fibroblast-like synoviocytes (SFCs) results in an increase of steady-state APN/CD13 mRNA and a rapid expression of cell-surface protein on the lymphocytes. In this study using the Dual-Luciferase reporter assay, we demonstrate that interaction of the T-cell line Jurkat with SFCs results in a higher activity of the APN/CD13 myeloid promoter in T cells. An enhancer located between the myeloid and epithelial APN/CD13 promoter increases the response of the promoter to the cell-cell contact-induced expression of APN/CD13 in lymphocytes. Adhesion of lymphocytes to extracellular matrix did not result in increased promoter activity. The lymphocytic promoter response induced by direct cell-cell contact with SFCs is not affected by mutations of a proximal promoter element (nucleotides -48 to -35), which has a possible functional role in the basal APN/CD13 gene expression in lymphocytes. Upregulated peptidase-promoter activity via cell-cell contact shown in this study for the first time is discussed as a general mechanism in peptidase induction.     

Increased expression of interleukin-8 and aminopeptidase N by cell-cell contact: interleukin-8 is resistant to degradation by aminopeptidase N/CD13

In rheumatic joints, high concentrations of interleukin-8 (IL-8) have been measured in synovial fluid and in pannus tissue. In both locations aminopeptidase N (APN)/CD13, an exopeptidase with reported activity towards IL-8 is also present. The surprising stability of IL-8 in the presence of an alleged IL-8-degrading peptidase prompted us to undertake the present study. Cocultivation of fibroblast-like synoviocytes (SFC) with T cells or with T lymphocytic cell membranes, or of T cells with SFC cell membranes, all resulted in increased IL-8 mRNA expression and IL-8 secretion into the medium, and an increase of APN expression on lymphocytes. IL-8 degradation was monitored by Western blots and HPLC. IL-8(72), as a partially processed form, was used throughout this study since it is abundant in tissues and has increased biological activity in comparison to IL-8(77). Thus its degradation/inactivation is considered of high biological significance. Whereas trypsin as a positive control rapidly degraded IL-8, we did not see any IL-8 degradation, either by a variety of soluble APNs, by leucine aminopeptidase or by APN expressed on the surface of SFC, or on ECV304 cells transfected with an APN expression vector. The much more sensitive HPLC technique resulted in negative results as well.     

Soluble CD26/dipeptidyl peptidase IV induces T cell proliferation through CD86 up-regulation on APCs.

CD26 is a T cell costimulatory molecule with dipeptidyl peptidase IV enzyme activity in its extracellular region. We have previously reported that the addition of soluble CD26 (sCD26) resulted in enhanced proliferation of peripheral blood T lymphocytes induced by the recall Ag, tetanus toxoid (TT). However, the mechanism involved in this immune enhancement has not yet been elucidated. In this paper, we demonstrate that the enhancing effect of sCD26 on TT-induced T cell proliferation occurred in the early stages of immune response. The cells directly affected by exogenously added sCD26 are the CD14-positive monocytes in the peripheral blood. Mannose-6 phosphate interfered with the uptake of sCD26 into monocytes, suggesting that mannose-6 phosphate/insulin-like growth factor II receptor plays a role in the transportation of sCD26 into monocytes. When sCD26 was added after Ag presentation had taken place, enhancement in TT-induced T cell proliferation was not observed. In addition, enhancement of TT-mediated T cell proliferation by sCD26 does not result from trimming of the MHC-bound peptide on the surface of monocytes. Importantly, we also showed that exogenously added sCD26 up-regulated the expression of the costimulatory molecule CD86 on monocytes through its dipeptidyl peptidase IV activity, and that this increased expression of CD86 was observed at both protein and mRNA level. Therefore, our findings suggest that sCD26 enhances T cell immune response to recall Ag via its direct effect on APCs.     

Immunoelectron microscopic demonstration of the membrane proteases aminopeptidase N/CD13 and dipeptidyl peptidase IV/CD26 in normal and neoplastic renal parenchymal tissues and cells

Aminopeptidase N (APN, CD13) and dipeptidyl peptidase IV (DPP IV, CD26) are transmembrane ectoenzymes occurring in a wide variety of cells. They are involved in tumour cell invasion and the formation of metastases. A basis for further information about these enzymes is the exact ultrastructural localization in normal and malignant cells. In this paper, we demonstrate the precise subcellular localization of the membrane peptidases APN and DPP IV on the cell surfaces in renal tissues, renal cell carcinoma, cultured renal parenchymal cells and cultured renal carcinoma cells. Using cryo-ultramicrotomy of weakly fixed tissues and cells in combination with indirect immunogold labelling, both membrane peptidases were detectable on the external cell surfaces. They showed different ultrastructural expression patterns. Both membrane peptidases were abundantly labelled on the external cell surfaces of human kidney proximal tubular cells. The expression pattern of APN/CD13 and DPP IV/CD26 in single labelling was confirmed by a successive double labelling technique. The immunolabelling of CD13 on cultured renal parenchymal cells showed a stronger expression then in cells in vivo, but CD26 could not be found. In renal cell cancer (mixed clear cell/chromophilic, poorly differentiated and clear cell type, moderately differentiated) CD13 and CD26 were labelled as in benign renal tissue, but CD26 appeared overexpressed. On the renal carcinoma cells Caki-1 and Caki-2, only one of the two peptidases could be found. CD13 was present non-homogeneously in Caki-1, where the enzyme appeared to form clusters. When CD26 on the cultured renal carcinoma cells Caki-2, is compared with renal proximal tubular cells and renal carcinoma cells in tissue sections, a reduced expression is observed. CD13 was not detected in Caki-2, and CD26 was not found in Caki-1. These small changes on the cell surfaces can only be detected by electronmicroscopic methods. The differences in the distribution of APN/CD13 and DPP IV/CD26 in normal and malignant cells are discussed in connection with literature. Further investigations, especially labelling studies on other neoplastic tissues and cells, will be necessary in order to explain the precise role these membrane peptidases in malignancies.     

Regulation of aminopeptidase-N (CD13) and Fc epsilon RIIb (CD23) expression by IL-4 depends on the stage of maturation of monocytes/macrophages.

IL-4 has multiple biologic activities and it has been shown to have effects on B and T lymphocytes, mast cells, NK cells, and monocytes. We studied the influence of IL-4 on the expression of cell membrane determinants, in particular aminopeptidase-N (CD13) and Fc epsilon RIIb (CD23), on human peripheral blood monocytes. We compared the response of monocytes with the response of human alveolar macrophages and monocytic cell lines (U937 and THP1), as mature and more immature representatives of the mononuclear phagocyte system, respectively. A dose-dependent increase of the expression of CD13 Ag was observed when monocytes were cultured with IL-4. Kinetic analyses revealed that this induction was maximal after 2 to 3 days of culture and resembled the kinetics of IL-4-induced expression of Fc epsilon RIIb on monocytes. This IL-4-induced increase was absent when monocytes were cultured with IL-4 and an anti-IL-4 antiserum. Concomitantly, an IL-4-induced increase in leucine-aminopeptidase activity could be observed. Northern blot analysis showed that incubation of monocytes with IL-4 induced a marked increase in CD13 mRNA. Alveolar macrophages also exhibited an increase in CD13 Ag expression when exposed to IL-4. Surprisingly, IL-4 was unable to induce expression of Fc epsilon RIIb on alveolar macrophages. U937 and THP1 cells did not show an induction of CD13 Ag when cultured in the presence of IL-4. However, IL-4 did induce the expression of Fc epsilon RIIb on both cell lines, suggesting the presence of functional IL-4R. Our data demonstrate that IL-4 increases the expression of CD13 Ag on monocytes. This IL-4-induced increase can also be observed in more mature monocytic cells such as alveolar macrophages, but is absent in immature cells such as U937 or THP1 cells. This is functionally accompanied by an increase in leucine-aminopeptidase activity and may be part of the general activation of monocytes/macrophages by IL-4. In conclusion, the data suggest that IL-4 responsiveness, in particular the induction of CD13 Ag and Fc epsilon RIIb expression, may be dependent on the stage of maturation of monocytes/macrophages.     

Aminopeptidase N (APN)/CD13-dependent CXCR4 downregulation is associated with diminished cell migration,proliferation and invasion

Aminopeptidase N (APN/CD13) is a 150 kDa membrane-bound ubiquitously expressed protease with a broad functional repertoire. It hydrolyzes small peptide mediators, modulates cell motility and adhesion to extracellular matrix and also acts as a viral receptor. In order to dissect the function of enzymatically active and inactive APN/CD13, substitutions of different enzymatic active amino acid residues were generated by site-directed mutagenesis and stably transfected into human embryonic kidney cells. All APN variants analyzed exhibited a complete loss of enzymatic activity, whereas wild type APN transfectants exerted a strong aminopeptidase-specific activity. Furthermore, wild type APN expression was associated with a significant decrease in proliferation, migration and also reduced anchorage-independent growth when compared to enzymatically inactive APN variants and controls. This appeared to be due to a downregulated mRNA and protein expression of the chemokine receptor CXCR4 and an inhibition of the stromal cell-derived factor (SDF)-1α/CXCL12-mediated migration. Thus, high APN enzyme activity may antagonize the cellular properties regulated by the CXCR4/SDF-1α system in embryonic kidney cells.     

Expression profile of FcgammaRIIb on leukocytes and its dysregulation in systemic lupus erythematosus

FcgammaRIIb (CD32B, Online Mendelian Inheritance in Man 604590), an IgG FcR with a tyrosine-based inhibitory motif, plays a critical role in the balance of tolerance and autoimmunity in murine models. However, the high degree of homology between FcgammaRIIb and FcgammaRIIa in humans and the lack of specific Abs to differentiate them have hampered study of the normal expression profile of FcgammaRIIb and its potential dysregulation in autoimmune diseases such as systemic lupus erythematosus (SLE). Using our newly developed anti-FcgammaRIIb mAb 4F5 which does not react with FcgammaRIIa, we found that FcgammaRIIb is expressed on the cell surface of circulating B lymphocytes, monocytes, neutrophils, myeloid dendritic cells (DCs), and at very low levels on plasmacytoid DCs from some donors. Normal donors with the less frequent 2B.4 promoter haplotype have higher FcgammaRIIb expression on monocytes, neutrophils, and myeloid DCs similar to that reported for B lymphocytes, indicating that FcgammaRIIb expression on both myeloid and lymphoid cells is regulated by the naturally occurring regulatory single nucleotide polymorphisms in the FCGR2B promoter. FcgammaRIIb expression in normal controls is up-regulated on memory B lymphocytes compared with naive B lymphocytes. In contrast, in active SLE, FcgammaRIIb is significantly down-regulated on both memory and plasma B lymphocytes compared with naive and memory/plasma B lymphocytes from normals. Similar down-regulation of FcgammaRIIb on myeloid-lineage cells in SLE was not seen. Our studies demonstrate the constitutive regulation of FcgammaRIIb by natural gene polymorphisms and the acquired dysregulation in SLE autoimmunity, which may identify opportunities for using this receptor as a therapeutic target.     

Circulating CD33+ large mononuclear cells contain three distinct populations with phenotype of putative antigen-presenting cells including myeloid dendritic cells and CD14+ monocytes with their CD16+ subset

BACKGROUND: In peripheral blood, myeloid markers identify a heterogeneous mixture of cells in transit from the bone marrow to peripheral tissues. Similarly, HLA-class II DR expression usually identifies mononuclear cells with the potential for developing antigen-presenting activity. We gathered putative antigen presenting cells bearing myeloid markers (My-APC) to study their composition by cell surface phenotype. METHODS: To gather and dissect My-APC phenotype while excluding lymphocytes and granulocytes, we developed a strategy based on staining red cell-lysed peripheral blood and gating cells bearing myeloid markers and physical parameters of large mononuclear cells. RESULTS: Phenotypic analysis within the My-APC gate showed three distinct populations. The largest fraction was constituted by CD14+ monocytes that extended into the other two populations, each expressing gradually lower levels of CD14 surface antigen along with increasing levels of CD16 and CD2, respectively. The CD16 and CD2 expression patterns extended from CD16+CD14+ or CD2+CD14+ double- positive intermediate cells toward each single positive subset, but they were reciprocally exclusive. Interestingly, CD2+CD14- cells within the My-APC gate were equivalent to myeloid dendritic cell precursors (pre-DC) defined previously by the absence of lineage markers and expression of HLA-DR and myeloid markers. Phenotypic analysis of each population revealed differences in the expression of costimulatory molecules and CD62L. CONCLUSIONS: This novel analytical approach allowed us to distinguish circulating My-APC in three subsets and to identify relationships between monocytes and other related myeloid populations including DC.     

Circulating CD2+ monocytes are dendritic cells

Low levels of CD2 have been described on subsets of monocytes, macrophages, and dendritic cells. CD2 is expressed on about one-third of circulating monocytes, at levels one-half log lower than on T or NK cells, representing 2-4% of PBMC. FACS analysis of CD2+ and CD2- monocytes revealed no significant difference in the expression of adhesion molecules (CD11a/b/c), class II Ags (HLA-DR, -DQ, -DP), myeloid Ags (CD13, CD14, CD33), or costimulatory molecules (CD80, CD86). Freshly isolated CD2+ and CD2- monocytes were morphologically indistinguishable by phase contrast microscopy. However, scanning electron microscopy revealed large prominent ruffles on CD2+ monocytes in contrast to small knob-like projections on CD2- monocytes. After 2 days of culture, the CD2+ monocytes largely lost CD14 expression and developed distinct dendrites, whereas the CD2- monocytes retained surface CD14 and remained round or oval. Freshly isolated CD2+ monocytes were more potent inducers of the allogeneic MLR and more efficiently induced proliferation of naive T cells in the presence of HIV-1 gp120 than did CD2- monocytes. After culture in the presence of GM/CSF and IL-4, CD2+ monocytes were up to 40-fold more potent than monocyte-derived dendritic cells or CD2- monocytes at inducing allogeneic T cell proliferation. These findings suggest that circulating CD2+ and CD2- monocytes are dendritic cells and the precursors of macrophages, respectively. Thus, dendritic cells are far more abundant in the blood than previously thought, and they and precursors of macrophages exist in the circulation as phenotypically, morphologically, and functionally distinct monocyte populations.     

MicroRNA‐125b modulates inflammatory chemokine CCL4 expression in immune cells and its reduction causes CCL4 increase with age

Chemokines play a pivotal role in regulating the immune response through a tightly controlled expression. Elevated levels of inflammatory chemokines commonly occur with aging but the mechanism underlying this age‐associated change is not fully understood. Here, we report the role of microRNA‐125b (miR‐125b) in regulating inflammatory CC chemokine 4 (CCL4) expression in human immune cells and its altered expression with aging. We first analyzed the mRNA level of CCL4 in eight different types of immune cells including CD4 and CD8 T‐cell subsets (naïve, central and effector memory), B cells and monocytes in blood from both young (≤42 years) and old (≥70 years) adults. We observed that monocytes and naïve CD8 T cells expressed higher levels of CCL4 and exhibited an age‐related increase in CCL4. We then found the level of miR‐125b was inversely correlated with the level of CCL4 in these cells, and the level of miR‐125b was reduced in monocytes and naïve CD8 T cells of the old compared to the young adults. Knock‐down of miR‐125b by shRNA in monocytes and naïve CD8 T cells led to an increase of CCL4 protein, whereas enhanced miR‐125b expression by transfection in naïve CD8 T cells resulted in a reduction of the CCL4 mRNA and protein in response to stimulation. Finally, we demonstrated that miR‐125b action requires the ‘seed’ sequence in 3′UTR of CCL4. Together these findings demonstrated that miR‐125b is a negative regulator of CCL4 and its reduction is partially responsible for the age‐related increase of CCL4.     

IL‐12 stimulates the osteoclast inhibitory peptide‐1 (OIP‐1/hSca) gene expression in CD4+ T cells

Immune cell products such as interferon (IFN)‐γ and interleukin (IL)‐12 are potent inhibitors of osteoclast formation. We previously characterized the human osteoclast inhibitory peptide‐1 (OIP‐1/hSca), a Ly‐6 gene family member and showed IFN‐γ modulation of OIP‐1 expression in bone marrow cells. Whether, IL‐12 regulates OIP‐1 expression in the bone microenvironment is unclear. Real‐time PCR analysis revealed that IL‐12 treatment significantly enhanced OIP‐1 mRNA expression in human bone marrow mononuclear cells. Because IL‐12 induces IFN‐γ production by T cells, we tested whether IFN‐γ participates in IL‐12 stimulation of OIP‐1 gene expression in these cells. IL‐12 treatment in the presence of IFN‐γ neutralizing antibody significantly increased OIP‐1 mRNA expression, suggesting that IL‐12 directly regulates OIP‐1 gene expression. Interestingly, real‐time PCR analysis demonstrated that IL‐12 induces OIP‐1 expression (3.2‐fold) in CD4+ T cells; however, there was no significant change in CD8+ T cells. Also, IL‐12 (10 ng/ml) treatment of Jurkat cells transfected with OIP‐1 gene (?1 to ?1,988 bp) promoter‐luciferase reporter plasmid demonstrated a 5‐fold and 2.7‐fold increase in OIP‐1 gene promoter activity in the presence and absence of antibody against IFN‐γ, respectively. We showed that STAT‐1,3 inhibitors treatment significantly decreased IL‐12 stimulated OIP‐1 promoter activity. Chromatin immunoprecipitation (ChIP) assay confirmed STAT‐3, but not STAT‐1 binding to the OIP‐1 gene promoter in response to IL‐12 stimulation. These results suggest that IL‐12 stimulates the OIP‐1 gene expression through STAT‐3 activation in CD4+ T cells. J. Cell. Biochem. 107: 104–111, 2009. © 2009 Wiley‐Liss, Inc.     

Triggering of the proteinase dipeptidyl peptidase IV (CD26) amplifies human T lymphocyte proliferation.

CD26 (Ta1, dipeptidyl peptidase IV) is a Mr 105,000 protein expressed at high levels on activated T lymphocytes and is a potential marker of memory T cells. Reciprocal immunodepletion and solid phase double determinant binding studies showed that mAb AC7 and the CD26-specific mAb anti-Ta1 reacted with spatially distinct sites on the same molecule. The proteinase dipeptidyl peptidase IV (DPP IV) was immunoprecipitated with mAb AC7 and its enzymatic activity directly assayed using an enzyme overlay membrane system. High levels of DPP IV activity were detected on the T cell tumor line CCRF-HSB-2 and on PBMC stimulated by a variety of methods. By itself, soluble mAb AC7 was not mitogenic for T cells but enhanced T cell proliferation that resulted from treatment with phorbol myristic acetate (PMA) in the presence of accessory cells. T cell proliferation was also induced by co-immobilized mAb AC7 and mAb OKT3 (anti-CD3). Cultures of T cells growing in the presence of IL-2 responded with accelerated growth when exposed to a combination of immobilized mAb AC7 and soluble mAb OKT3, a result not seen with freshly isolated T cells.