Objective To investigate how culture duration affects the expression of immune membrane proteins in human monocyte-derived dendritic cells (DCs) and their exosomes (DEXs). Methods Human monocytes were induced with recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) to differentiate into DCs and were subsequently matured with tumor necrosis factor α(TNF-α). Exosomes were isolated by ultracentrifugation, and DEXs were identified by transmission electron microscopy and Amnis imaging flow cytometry, which were also used to quantify the expression of immune membrane proteins on DCs and DEXs. Results On the 10th day of culture, DCs displayed high surface expression of CD11c, CD80, CD86, major histocompatibility complex class I (MHC-I), and MHC-II. Expression peaked at day 18(CD11c: 78.66%±20.33%, CD80: 76.41%±10.02%, CD86: 96.43%±0.43%, MHC-I: 84.71%±2.96%, MHC-II: 80.01%±7.03%). After day 24, the overall expression showed a declining trend, with statistically significant differences observed for all markers except CD80 and MHC-II. By day 30, 80% of the DCs still expressed CD80, CD86, and MHC-II. The expression of immune membrane proteins on DEX surfaces also reached its peak on day 18, followed by an overall decline with prolonged culture time, with statistically significant differences observed for all markers except CD80. Correlation analysis revealed a significant positive relationship between the expression levels of immune membrane proteins on DC and DEX surfaces (CD11c: r=0.98; CD80: r=0.65; CD86: r=0.82; MHC-I: r=0.86; MHC-II: r=0.93). Conclusion Human monocyte-derived DCs in vitro express high expression of immune membrane proteins and maintain stable expression over a specific period. The exosomes secreted by these cells similarly demonstrate high surface expression of immune membrane proteins, with temporal trends aligned with those of the parent DCs.
Humans ; Dendritic Cells/immunology* ; Exosomes/immunology* ; Monocytes/metabolism* ; Cells, Cultured ; Time Factors ; B7-1 Antigen/metabolism* ; Membrane Proteins/immunology* ; Cell Culture Techniques/methods* ; B7-2 Antigen/metabolism* ; Cell Differentiation ; CD11c Antigen/metabolism* ; Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology*

Differentiated HL-60 is an effector cell widely used for the opsonophagocytic-killing assay (OPKA) to measure efficacy of pneumococcal vaccines. We investigated the correlation between phenotypic expression of immunoreceptors and phagocytic ability of HL-60 cells differentiated with N,N-dimethylformamide (DMF), all-trans retinoic acid (ATRA), or 1alpha, 25-dihydroxyvitamin D3 (VitD3) for 5 days. Phenotypic change was examined by flow cytometry with specific antibodies to CD11c, CD14, CD18, CD32, and CD64. Apoptosis was determined by flow cytometry using 7-aminoactinomycin D. Function was evaluated by a standard OPKA against serotype 19F and chemiluminescence-based respiratory burst assay. The expression of CD11c and CD14 gradually increased upon exposure to all three agents, while CD14 expression increased abruptly after VitD3. The expression of CD18, CD32, and CD64 increased during differentiation with all three agents. Apoptosis remained less than 10% until day 3 but increased after differentiation by DMF or ATRA. Differentiation with ATRA or VitD3 increased the respiratory burst after day 4. DMF differentiation showed a high OPKA titer at day 1 which sustained thereafter while ATRA or VitD3-differentiated cells gradually increased. Pearson analysis between the phenotypic changes and OPKA titers suggests that CD11c might be a useful differentiation marker for HL-60 cells for use in pneumococcal OPKA.
Antibodies, Bacterial/immunology ; Antigens, CD11c/metabolism ; Antigens, CD14/metabolism ; Antigens, CD18/metabolism ; Apoptosis/*immunology ; Biological Assay ; Cell Differentiation ; Cell Line, Tumor ; Cholecalciferol/pharmacology ; Dimethylformamide/pharmacology ; Flow Cytometry ; HL-60 Cells ; Humans ; Phagocytosis/*immunology ; Pneumococcal Vaccines/*immunology ; Receptors, IgG/metabolism ; Receptors, Immunologic/*biosynthesis ; Respiratory Burst/immunology ; Streptococcus pneumoniae/*immunology ; Tretinoin/pharmacology

Differentiated HL-60 is an effector cell widely used for the opsonophagocytic-killing assay (OPKA) to measure efficacy of pneumococcal vaccines. We investigated the correlation between phenotypic expression of immunoreceptors and phagocytic ability of HL-60 cells differentiated with N,N-dimethylformamide (DMF), all-trans retinoic acid (ATRA), or 1alpha, 25-dihydroxyvitamin D3 (VitD3) for 5 days. Phenotypic change was examined by flow cytometry with specific antibodies to CD11c, CD14, CD18, CD32, and CD64. Apoptosis was determined by flow cytometry using 7-aminoactinomycin D. Function was evaluated by a standard OPKA against serotype 19F and chemiluminescence-based respiratory burst assay. The expression of CD11c and CD14 gradually increased upon exposure to all three agents, while CD14 expression increased abruptly after VitD3. The expression of CD18, CD32, and CD64 increased during differentiation with all three agents. Apoptosis remained less than 10% until day 3 but increased after differentiation by DMF or ATRA. Differentiation with ATRA or VitD3 increased the respiratory burst after day 4. DMF differentiation showed a high OPKA titer at day 1 which sustained thereafter while ATRA or VitD3-differentiated cells gradually increased. Pearson analysis between the phenotypic changes and OPKA titers suggests that CD11c might be a useful differentiation marker for HL-60 cells for use in pneumococcal OPKA.
Antibodies, Bacterial/immunology ; Antigens, CD11c/metabolism ; Antigens, CD14/metabolism ; Antigens, CD18/metabolism ; Apoptosis/*immunology ; Biological Assay ; Cell Differentiation ; Cell Line, Tumor ; Cholecalciferol/pharmacology ; Dimethylformamide/pharmacology ; Flow Cytometry ; HL-60 Cells ; Humans ; Phagocytosis/*immunology ; Pneumococcal Vaccines/*immunology ; Receptors, IgG/metabolism ; Receptors, Immunologic/*biosynthesis ; Respiratory Burst/immunology ; Streptococcus pneumoniae/*immunology ; Tretinoin/pharmacology

Dendritic cells have been known as a member of strong innate immune cells against infectious organelles. In this study, we evaluated the cytokine expression of splenic dendritic cells in chronic mouse toxoplasmosis by tissue cyst-forming Me49 strain and demonstrated the distribution of lymphoid dendritic cells by fluorescence-activated cell sorter (FACS). Pro-inflammatory cytokines, such as IL-1alpha, IL-1beta, IL-6, and IL-10 increased rapidly at week 1 post-infection (PI) and peaked at week 3 PI. Serum IL-10 level followed the similar patterns. FACS analysis showed that the number of CD8alpha+/CD11c+ splenic dendritic cells increased at week 1 and peaked at week 3 PI. In conclusion, mouse splenic dendritic cells showed early and rapid cytokine changes and may have important protective roles in early phases of murine toxoplasmosis.
Animals ; Antigens, CD11c/analysis ; Antigens, CD8/analysis ; Cytokines/*blood/*secretion ; Dendritic Cells/chemistry/*immunology ; Disease Models, Animal ; Flow Cytometry ; Mice ; Mice, Inbred BALB C ; Rodent Diseases/immunology ; Spleen/*immunology ; Time Factors ; Toxoplasmosis, Animal/*immunology

Secondary lymphoid tissue chemokine (SLC), which is expressed in T cell zones of secondary lymphoid organs, including the spleen and lymph nodes, strongly recruits both T lymphocytes and mature dendritic cells. As appropriate interaction of tumor-specific T cells and mature dendritic cells, equipped with tumor antigens, is a prerequisite for effective T cell immunity against established tumors, we mobilized lymphocytes and dendritic cells to tumor sites by intratumoral injection of secondary lymphoid tissue chemokine-Fc (SLC-Fc) fusion protein using the B16F10 murine melanoma model. Activation of dendritic cells, another prerequisite for the effective activation of naive tumor-specific T cells, was achieved by the addition of immunostimulatory cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG-ODN) into the tumor site. Intratumoral administration of SLC-Fc or CpG-ODN revealed antitumor effects against B16F10 murine melanoma grown in the subcutaneous space. Co-treatment of SLC-Fc and CpG-ODN displayed synergistic effects in reducing the tumor size. The synergistic antitumor effect in co-treatment group was correlated with the synergistic/additive increase in the infiltration of CD4+ T cells and CD11c+ dendritic cells in the tumor mass compared to the single treatment groups. These results suggest that the combined use of chemokines and adjuvant molecules may be a possible strategy in clinical tumor immunotherapy.
Animals ; Antigens, CD11c/immunology ; CD4-Positive T-Lymphocytes/immunology ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Chemokine CCL21/*administration & dosage/pharmacology ; Chemotaxis, Leukocyte ; Dendritic Cells/immunology/metabolism ; Immunotherapy ; Injections, Intralesional ; Melanoma, Experimental/*immunology/*therapy ; Mice ; Mice, Inbred C57BL ; Oligodeoxyribonucleotides/*administration & dosage/pharmacology ; T-Lymphocytes/immunology/metabolism

OBJECTIVETo examine the in vivo anti-metastatic effect of enhanced expression of CD40L cDNA in murine ovarian cancer OVHM cells (CD40L-OVHM) injected into the spleen on liver metastasis in mice.

METHODSOVHM cells were inoculated into the spleen of 6 to 8 week-old female B6C3F1 (C57BL/6N x C3H/He) mice. The established liver metastasis was identified by histopathology (HE staining). OVHM cells, DNA-pMKITneo-OVHM cells or CD40L-OVHM cells were inoculated into the spleen of female B6C3F1 mice and the expressions of CD11c in splenic cells were detected by flow cytometry. The specific cytotoxicity of splenic cells was detected by MTT assay, and the serum cytokines of IFN-gamma, TNF-alpha, IL-12, IL-4 and IL-10 of the mice were measured by enzyme linked immunoabsorbent assay. The liver metastases and the survival time of the mice were also recorded.

RESULTSThe mouse models with liver metastasis by injecting tumor cells into the spleen of mice were established. The expression of CD11c and the specific killing rate in CD40L-OVHM cells group was significantly higher than that in the OVHM cells group and DNA-pMKITneo-OVHM cells group. The expressions of IFN-gamma, TNF-alpha and IL-12 in the CD40L-OVHM cells group were much more increased than OVHM cells group and DNA-pMKITneo-OVHM cells group, but the expressions of IL-4 and IL-10 in the CD40L-OVHM cells group were decreased significantly (p < 0.05). The average weights of livers and spleens of mice in CD40L-OVHM cells group were significantly lower than those of DNA-pMKITneo-OVHM cells group and OVHM cells group. The survival time of mice in CD40L-OVHM cells group was also significantly longer than that in the OVHM cells group and DNA-pMKITneo-OVHM cells group (P < 0.05).

CONCLUSIONThe data directly demonstrate that the expression of CD40L in ovarian cancer cells (CD40L-OVHM) can enhance the proliferation and differentiation of dendritic cells in the spleen and induce specific cytotoxic effect of T cells in the spleen, and may regulate the immune function of peripheral blood cells and the immune balance between Th1 cells and Th2 cells, which maybe the possible mechanism induced by CD40L in mice inhibiting the development of liver metastasis.

Animals ; CD11c Antigen ; metabolism ; CD40 Ligand ; genetics ; metabolism ; Cell Differentiation ; Cell Line, Tumor ; Cell Proliferation ; DNA, Complementary ; genetics ; Dendritic Cells ; cytology ; Female ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-12 ; metabolism ; Interleukin-4 ; metabolism ; Liver Neoplasms ; pathology ; secondary ; Mice ; Mice, Inbred C3H ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Ovarian Neoplasms ; metabolism ; pathology ; Spleen ; metabolism ; T-Lymphocytes, Cytotoxic ; immunology ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo investigate the clinicopathologic features, diagnosis, differential diagnosis and the prognosis of hairy cell leukemia (HCL).

METHODSFifteen splenectomy specimens of HCL patients were investigated retrospectively using HE and immunohistochemistry in correlation with the follow-up information.

RESULTS(1) The male to female ratio was 2.75:1, age ranged from 36 to 68 years with a median of 47 years. The most consistent clinical feature at presentation was marked splenomegaly (100%). Other symptoms included anemia (80.0%), thrombocytopenia (60.0%), leucocytosis (53.3%), pancytopenia (20.0%) and the absence of B-symptom. (2) The proportion of hairy cells was (14.6 +/- 7.2)% in periphery blood and (47.3 +/- 23.8)% in bone marrow. The positive rate of TRAP assay was 62.5% in bone marrow; 85.7% for TPA test and the detection rate for RLC was 25% by transmission electric microscopy. The frequency of bone marrow involvement was 100%. (3) The average weight of 15 spleens was (3012 +/- 1974) g. The size of 6 spleens ranged from 16 cm x 10 cm x 5 cm to 32 cm x 20 cm x 14 cm. The white pulp of spleen showed a characteristic atrophy feature or even absent due to leukemic infiltration, predominantly involving the red pulp with some sinusoidal pattern. "Blood pool" change was an infrequent feature (3/15 cases). The nuclei of leukemic cells were round (13 cases) or bean-shaped (2 cases), nucleoli inconspicuous or disappeared. The abundant cytoplasm and prominent cell border resulted in a "fried egg" appearance. By immunohistochemistry, leukemic cells were positive for CD45RA, CD20, PAX-5, CD25, CD11c, Annexin A1 and cyclinD1, but negative for CD3 and CD43. (4) 13 cases (86.7%) have been followed-up and all are alive. Among them, 9 cases are living well more than 5 years and 7 more than 10 years.

CONCLUSIONSSplenomegaly is frequently the first manifestation of patients with HCL and occurred predominantly in the middle to elderly adults. Definite diagnosis of HCL requires a combined histological and immunohistochemical assessment of the splenectomy specimen, bone marrow biopsy and aspirate.

Adult ; Aged ; Annexin A1 ; metabolism ; Antigens, CD20 ; metabolism ; CD11c Antigen ; metabolism ; CD79 Antigens ; metabolism ; Diagnosis, Differential ; Female ; Follow-Up Studies ; Humans ; Ki-67 Antigen ; metabolism ; Leukemia, Hairy Cell ; metabolism ; pathology ; surgery ; Leukemia, Lymphocytic, Chronic, B-Cell ; metabolism ; pathology ; Leukemia, Prolymphocytic ; metabolism ; pathology ; Leukocyte Common Antigens ; metabolism ; Lymphoma, B-Cell, Marginal Zone ; metabolism ; pathology ; Lymphoma, Follicular ; metabolism ; pathology ; Lymphoma, Mantle-Cell ; metabolism ; pathology ; Male ; Middle Aged ; Retrospective Studies ; Spleen ; pathology ; Splenectomy ; Survival Rate

OBJECTIVETo investigate the effects of CDA-II alone or combined with cAMP on the retinoic acid (RA)-resistant acute promyelocytic leukemia (APL) cells.

METHODSThe RA-resistant cell line NB4-R2 was used as an in vitro model and treated with CDA-II alone or in combination with cAMP. Cell apoptosis was assessed by morphology observation, distribution of cellular DNA contents and sub-G1 cell population. The level of Bcl-2 was detected by flow cytometry, DNA "ladder" was detected by agarose-electrophoresis.

RESULTSCDA-II could induce NB4-R2 cell apoptosis through decreasing the level of cellular anti-apoptotic protein Bcl-2. cAMP could significantly enhance the role of CDA-II. Bcl-2 positive cell rates decreased to (15.1 +/- 4.8)% and (7.3 +/- 2.9)% in NB4-R2 cells treated with 1 mg/ml CDA-II plus 100 micromol/L cAMP for 48 h and 72 h, respectively. While 100 micromol/L of cAMP could decrease Bcl-2 positive NB4-R2 cells from (92.0 +/- 0.6)% to (75.3 +/- 2.0)%.

CONCLUSIONSCDA-II combined with cAMP could exert potent apoptotic effect on RA-resistant APL cells.

Animals ; Apoptosis ; drug effects ; CD11c Antigen ; metabolism ; Cells, Cultured ; Cyclic AMP ; pharmacology ; Humans ; Leukemia, Promyelocytic, Acute ; drug therapy ; pathology ; Peptides ; pharmacology ; Phenylacetates ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Tretinoin ; pharmacology

OBJECTIVETo investigate the effects of mobile phone 1800 MHz electromagnetic fields (EMF) on the surface markers and the functions of human dendritic cells (DC).

METHODSHuman DCs were exposed to intermittent 5 min on/10 min off EMF with specific absorption rates (SAR) 4 W/kg for 0 h, 1 h, 12 h or 24 h, respectively. FACS analysis was used to detect the positive percentage of DC surface markers including HLA-DR and co-stimulatory molecules such as CD80, CD86, CD40 and CD11c. CCK-8 kit was adopted to examine the function of allo-mixed lymphocyte reaction (allo-MLR) of DC, and enzyme linked immunosorbent assay (ELISA) to identify the levels of IL-12p70 and TNF-alpha secreted by DC.

RESULTCompared with the sham radiation group, after exposure to the electromagnetic fields for 1 h, 12 h, or 24 h, HLA-DR, CD80,CD86 and CD40 were all declined except CD11c. The ability of DC allo-MLR in each exposure group was decreased significantly (P<0.05), especially in the 24 h exposure group. However, the secreted levels of IL-12p70 and TNF-alpha of DC in each exposure group remained no changed.

CONCLUSIONThe study showed that EMF exposure could down-regulate the surface molecules and stimulation ability of human DC.

B7-1 Antigen ; B7-2 Antigen ; immunology ; Biomarkers ; analysis ; CD11c Antigen ; immunology ; Cell Phone ; Cells, Cultured ; Dendrites ; pathology ; Dendritic Cells ; metabolism ; physiology ; radiation effects ; Electromagnetic Fields ; HLA-DR Antigens ; analysis ; Humans ; Interleukin-12 ; immunology

Adult ; CD11c Antigen ; Case-Control Studies ; Dendritic Cells ; cytology ; metabolism ; Female ; Hepatitis B virus ; Hepatitis B, Chronic ; immunology ; metabolism ; Humans ; Male ; Middle Aged