Phenotypic and functional characteristics of spermatogonial stem cells in rats

Spermatogonial stem cells (SSCs) are at the foundation of the highly productive spermatogenic process that continuously produces male gametes throughout postnatal life. However, experimental evaluation of SSCs in postnatal testes is complicated because these cells are extremely rare and few defining morphology or biochemical characteristics are known. In this study, we used the spermatogonial transplantation functional assay, combined with fluorescence-activated cell sorting (FACS) analysis to identify cellular, biochemical and surface antigenic characteristics of SSCs in rat testes during development. Our results demonstrated that forward scatter (FSc)(hi), side scatter (SSc)(hi), mitochondria membrane potential (DeltaPsim)(lo), Ep-CAM(+), Thy-1(+), beta3-integrin(+) stem cells in neonate rat testes become SSc(lo), DeltaPsim(hi), Ep-CAM(+), Thy-1(lo), beta3-integrin(-) stem cells in pup rat testes. Furthermore, prospective identification of rat testis cell populations (Ep-CAM(+)), highly enriched for SSCs (1 in 13 for neonate; 1 in 8.5 for pup) enabled us to predict the Thy-1 and beta3-integrin status of stem cells in neonate and pup testes, which was subsequently confirmed by transplantation analyses. Systematic characterization of SSCs enabled the production of testis cell populations highly enriched (up to 120-fold) for SSCs and will facilitate future investigations of functional and genomic characteristics.     

Phenotypic and functional characteristics of porcine peritoneal mesothelial cells

The objective of this study was to establish a technique to isolate porcine mesothelial cells (PMCs) from omental tissue and to compare them to human mesothelial cells (HMCs). The PMCs were dispersed by collagenase digestion and isolated on a Ficoll layer. Their morphologic and ultrastructural features were assessed at confluence by light and electronic microscopy, and they were characterized by immunohistochemistry using specific HMC markers. PMC proliferation was studied in the presence of growth factors platelet-derived growth factor (PDGF), epidermal growth factor (EGF) or transforming growth factors beta1, beta2, or beta3 (TGF). Fibrinolytic PMC activity was detected by zymography for tissue plasminogen activator (tPA) and by reverse zymography for plasminogen activator inhibitor-1 (PAI-1). The recalcification time of cell lysates was used to define PMC procoagulant activity, and gelatinase zymography was used to detect metalloproteinase production. At confluence, PMCs formed typical cobblestone monolayers and exhibited structural features characteristic of HMCs. Weibel Palade bodies were never seen. Specific HMC markers (HBME1, ME1, WT1) cross-reacted with PMCs. As HMCs and PMCs coexpressed cytokeratin and vimentin, and also expressed vinculin and alpha-actin. Addition of PDGF or EGF to the culture medium stimulated PMC proliferation. PMCs constitutively expressed fibrinolytic and procoagulant activity and secreted MMP9 and MMP2. The technique described in this study allows isolation of mesothelial cells from porcine omental tissue. These porcine cells exhibit a mesothelial phenotype and functional properties similar to those of HMCs. Our data warrant an evaluation of mesothelial cells as targets in several therapeutic strategies with porcine models.     

Phenotypic and functional characteristics of macrophage-like cells differentiated in pro-inflammatory cytokine-containing cultures

Previous studies have demonstrated an infiltration of monocytes and increased levels of IL-1beta and TNF-alpha in some chronic inflammatory tissues. Interleukin-1beta and TNF-alpha are capable of protecting monocytes from spontaneous apoptosis and thus maintain their viability in vitro. To study the possible effects of these cytokines on the differentiation and function of recruited monocytes, a model has been developed in which monocytes isolated from human peripheral blood were differentiated into macrophages in serum in the presence or absence of IL-1beta or TNF-alpha. Monocytes cultured with IL-1beta and TNF-alpha underwent substantial changes in morphology, similar to those observed in monocytes undergoing differentiation into macrophages. The cultured cells increased in size and vacuolization and their content of acid phosphates increased 10-fold. Although they exhibited the morphological characteristics of macrophages, monocytes matured in the cytokines differed functionally from those cultured in serum in a lower expression of HLA-DR, lower ability for triggering the proliferation of allogeneic lymphocytes, higher expression of mannose receptor and greater production of superoxide and TNF-alpha. This data suggests that IL-1beta and TNF-alpha direct monocyte differentiation into macrophages with a reduced antigen-presenting and an increased pro-inflammatory factor-releasing phenotype. Elevated levels of IL-1beta and TNF-alpha in the inflammatory tissues may therefore not only prolong the survival of recruited monocytes, but maintain them in an inflammatory state.     

Phenotypic and functional characteristics of human newborns' B lymphocytes.

It has been demonstrated two major facts concerning human newborns' B lymphocytes: 1) they differentiate poorly into Ig-producing cells and 2) they express CD5 and CD1c membrane proteins. We have further analyzed human newborns' B cell characteristics and found that approximately half of them express activation Ag, i.e., 4F2 and IL-2R, both associated in significant proportions with CD23 and Bac-1. These membrane Ag were found both on CD5(+) and CD5(-) B cells. Newborns' B cells do not exhibit other activation markers because they express surface IgD, and because their size, RNA, and DNA contents do not differ from those of adults' B cells, indicating that they are in the G0/G1 cell cycle phase. Newborns' B cell proliferation can be induced by rIL-2, rIL-4, low m.w. B cell growth factor, and by Staphylococcus aureus protein A. It is presently difficult to build a hypothesis accounting for all the specific findings made on newborns' B cells. It is not known for instance whether CD5(+) and (-) B cells belong to distinct subsets as suggested by the fluorescence intensity curve obtained with an anti-CD5 antibody or to distinct stages in a unique pattern of B cell maturation during fetal and newborn life. This may indicate that partially activated B cells actually produce natural polyspecific autoantibodies of the IgM isotype found in newborns' human serum.     

Maturational steps of bone marrow-derived dendritic murine epidermal cells. Phenotypic and functional studies on Langerhans cells and Thy-1+ dendritic epidermal cells in the perinatal period

The adult murine epidermis harbors two separate CD45+ bone marrow (BM)-derived dendritic cell systems, i.e., Ia+, ADPase+, Thy-1-, CD3- Langerhans cells (LC) and Ia-, ADPase-, Thy-1+, CD3+ dendritic epidermal T cells (DETC). To clarify whether the maturation of these cells from their ill-defined precursors is already accomplished before their entry into the epidermis or, alternatively, whether a specific epidermal milieu is required for the expression of their antigenic determinants, we studied the ontogeny of CD45+ epidermal cells (EC). In the fetal life, there exists a considerable number of CD45+, Ia-, ADPase+ dendritic epidermal cells. When cultured, these cells become Ia+ and, in parallel, acquire the potential of stimulating allogeneic T cell proliferation. These results imply that CD45+, Ia-, ADPase+ fetal dendritic epidermal cells are immature LC precursors and suggest that the epidermis plays a decisive role in LC maturation. The day 17 fetal epidermis also contains a small population of CD45+, Thy-1+, ADPase-, CD3- round cells. Over the course of 2 to 3 wk, they are slowly replaced by an ever increasing number of round and, finally, dendritic CD45+, Thy-1+, CD3+ EC. Thus, CD45+, Thy-1+, ADPase-, CD3- fetal EC may either be DETC precursors or, alternatively, may represent a distinctive cell system of unknown maturation potential. According to this latter theory, these cells would be eventually outnumbered by newly immigrating CD45+, Thy-1+, CD3+ T cells--the actual DETC.     

Phenotypic characteristics of heat-resistant murine neuroblastoma cells

One-step selection of murine neuroblastoma (N18TG2 cell line) for the resistance to injuring action of higher temperature resulted in obtaining cell line NTR1 stably capable of proliferating at 40 degrees C. The thermoresistance is shown to be coupled with the changes in some phenotypic features of NTR1 cells: the multiplication rate, saturation density of cells, morphological features, inducibility of long neurite-like processes and adhesion. A possible significance of plasma membrane changes in genetically stable thermoresistance of NTR1 neuroblastoma cells is discussed.     

Phenotypic characteristics of natural killer cells in acute hepatitis

Natural killer (NK) cells are the principal effector cell population in innate immune defense against many types of infections. These cells are enriched in the liver, where they comprise approximately 40% to 60% of the intrahepatic lymphocyte pool compared to the peripheral blood compartment. In chronic HBV and HCV infection, NK cells were reported to be partially dysfunctional due to impaired IFN-Γ secretion. Few studies have examined phenotypic features of NK cells in acute hepatitis. We identified NK (CD56+CD3-) cell populations in patients with acute hepatitis by examining the expressions of phenotypic NK cell markers (CD16, NKG2A, and NKG2D). Peripheral blood mononuclear cells were isolated from patients with acute hepatitis A (7) and patients with non-viral acute toxic hepatitis (6) during the symptomatic and convalescent phases. Expressions of NK (CD56+CD3-) cell markers, CD16, NKG2A, and NKG2D, were measured by flow cytometry. Symptomatic acute hepatitis including non-viral hepatitis and HAV infection showed significant increases of NKG2A expression compared to healthy controls. Interestingly, there was a direct correlation between the proportion of NK cell populations and liver function parameters (AST, ALT) in HAV infection. The strong correlation was also observed between the expression of NKG2A+NK cells and ALT, which suggests that most of NK cells in severe phase of disease express high level of NKG2A on their surface. In addition, decreased number of NK cells (CD56+CD3-) in symptomatic phase began to increase in the convalescent phase of acute hepatitis A. However, the expression of NKG2A tended to be reduced, which indicates that NKG2A, the inhibitory receptor on NK cells, can be a severity parameter in acute hepatitis.     

Phenotypic and functional characterization of ultraviolet radiation-induced regulatory T cells

Sensitization through UV-exposed skin induces regulatory T cells (Treg). In contrast to the classical CD4+CD25+ Treg that act contact dependent, UV-induced Treg (UV-Treg) suppress via IL-10, indicating a distinct subtype that requires further characterization. Depletion studies revealed that UV-Treg express the glucocorticoid-induced TNF family-related receptor (GITR) and the surface molecule neuropilin-1. The injection of T cells from UV-tolerized mice after depletion of UV-Treg into naive recipients enabled a contact hypersensitivity response, indicating that tolerization also induces T effector cells. Adoptive transfer experiments using IL-10-deficient mice indicated that the IL-10 required for suppression is derived from UV-Treg and not from host-derived cells. Activation of UV-Treg is Ag specific, however, once activated suppression is nonspecific (bystander suppression). Hence, speculations exist about the therapeutic potential of Treg generated in response to Ag that are not necessarily the precise Ag driving the pathogenic process. Thus, we studied the consequences of multiple injections of 2,4-dintrofluorobenzene (DNFB)-specific Treg into ears of naive mice followed by multiple DNFB challenges. DNFB-specific Treg were injected once weekly into the left ears of naive mice and DNFB challenge was performed always 24 h later. After three injections, a challenging dose of DNFB was applied on the right ear. This resulted in pronounced ear swelling, indicating that the subsequent boosting of DNFB-specific Treg had caused sensitization of the naive mice against DNFB. These data demonstrate that UV-Treg express GITR and neuropilin-1 and act via bystander suppression. However, constant boosting of Treg with Ag doses in the challenging range results in final sensitization that might limit their therapeutic potential.     

Phenotypic and functional analysis of EBV-specific memory CD8 cells in SLE

T cell dysfunction has been described in systemic lupus erythematosus (SLE). However, the specific phenotype and function of antigen-specific CD8 cells is less clear. Here we determined phenotype and function of Epstein-Barr virus (EBV)-specific CD8 cells at the single-cell level in SLE. HLA-A2-restricted EBV-BMLF-1-specific CD8 cells were enumerated by flow cytometry using tetramers in SLE and healthy control subjects. Antigen-specific CD8 cells were analyzed for expression of differentiation, activation, proliferation, and anti-apoptotic markers. EBV-specific, other virus-specific (specific against a viral peptide pool consisting of cytomegalovirus, EBV and influenza virus peptides), and mitogen-induced CD8 cell function was assessed by INF-gamma ELISPOT assay. Frequencies of EBV-specific CD8 cells tended to be greater in SLE subjects than in healthy control subjects (p=0.07). While over 10% of EBV-specific CD8 cells were capable of producing IFN-gamma in four out of five healthy control subjects, such proportions of EBV-specific CD8 cells capable of IFN-gamma production were observed in only one out of six SLE subjects (p=0.04). In contrast, viral peptide pool-specific and mitogen-induced IFN-gamma-producing T cell function was intact in SLE subjects. Phenotypic analysis revealed EBV-specific CD8 cells to be in an early to intermediate differentiation and resting memory state in both groups. While EBV-specific CD8 cells are similar in phenotype, their frequency tends to be increased, and function appears to be decreased in SLE. Therefore, an impaired EBV-specific CD8 immune response may exist in SLE, potentially contributing to disease pathogenesis.     

Phenotypic and functional maturation of dendritic cells mediated by heparan sulfate

Primary immune responses are thought to be induced by dendritic cells. To promote such responses, dendritic cells must be activated by exogenous agonists, such as LPS, or by products of activated leukocytes, such as TNF-alpha and IL-1. How dendritic cells might be activated in the absence of exogenous stimuli, or without the immediate presence of activated leukocytes, as might occur in immunity to tumor cells or transplants, is unknown. We postulated that heparan sulfate, an acidic, biologically active polysaccharide associated with cell membranes and extracellular matrices, which is rapidly released under conditions of inflammation and tissue damage, might provide such a stimulus. Incubation of immature murine dendritic cells with heparan sulfate induced phenotypic maturation evidenced by up-regulation of I-A, CD40, CD54 (ICAM-1), CD80 (B7-1), and CD86 (B7-2). Dendritic cells exposed to heparan sulfate exhibited a markedly lowered rate of Ag uptake and increased allostimulatory capacity. Stimulation of dendritic cells with heparan sulfate induced release of TNF-alpha, IL-1beta, and IL-6, although the maturation of dendritic cells was independent of these cytokines. These results suggest that soluble heparan sulfate chains, as products of the degradation of heparan sulfate proteoglycan, might induce maturation of dendritic cells without exogenous stimuli, thus contributing to the generation and maintenance of primary immune responses.     

Phenotypic and functional evaluation of suppressor cells in normal pregnancy and in chronic aborters

To evaluate the potential role of immunoregulatory cells modulating the maternal immunologic response during pregnancy, we carried out phenotypic and functional studies in patients with normal obstetrical histories during each trimester and in patients with chronic idiopathic spontaneous abortions. Using monoclonal antibodies (Ortho), total numbers of T cells (T3+) and T4+ cells progressively increased during pregnancy (compared to nonpregnant controls) and then declined in the third trimester. Increased percentages of T8+, T10+, and Ia+ cells were found in the third trimester. The relative decline in numbers of T4+ cells, with increased numbers of T8+ cells, led to a significantly reduced T4/T8 ratio in the third trimester. Histamine receptors on T cells were quantitated by an immunofluorescent technique. Significantly reduced numbers of H1-type receptors were noted during the second trimester of pregnancy and this was associated with a decreased H1/H2 ratio. Functionally, histamine-induced suppression was measured in a lymphocyte proliferation assay. Patients in the first and second trimester of pregnancy had greater histamine-induced suppression of phytohemagglutinin (PHA)-stimulated proliferation at high concentrations of histamine (10(-3) to 10(-7)) but less suppression at the lower concentrations (10(-9) to 10(-11) M), compared to nonpregnant controls. In contrast, patients studied in the third trimester failed to respond to any concentration of histamine. MLC-induced suppressor activity was generated by incubating the maternal cells with either paternal or third-party mononuclear cells for 2 or 6 days and assaying the cell-free supernatant for its suppressive effects on PHA-stimulated proliferation. Maternal responses to paternal cells did not result in significant suppression in 2-day supernatants during any trimester but by 6 days the suppressive activity was equivalent to non-pregnant controls in patients during the first and second trimester. Maternal responses to third party cells was greater during the second trimester than either the first or third trimesters in both 2- and 6-day supernatants. Patients with histories of chronic idiopathic spontaneous abortions, who were not pregnant at the time of study, exhibited normal numbers of T-cell subsets and T4/T8 ratios. Numbers of both H1 and H2 receptor bearing T cells were proportionally reduced, resulting in a normal H1/H2 ratio. Despite having decreased numbers of H1 and H2 receptor bearing cells, histamine-induced suppression of PHA-stimulated proliferation was comparable to nonpregnant controls over the concentration range (10(-3) to 10(-11) M) employed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phenotypic distinction and functional characterization of pro-B cells in adult mouse bone marrow

A lymphoid-committed progenitor population was isolated from mouse bone marrow based on the cell surface phenotype Thy-1.1(neg)Sca-1(pos)c-Kit(low)Lin(neg). These cells were CD43(pos)CD24(pos) on isolation and proliferated in response to the cytokine combination of steel factor, IL-7, and Flt3 ligand. Lymphoid-committed progenitors could be segregated into more primitive and more differentiated subsets based on expression of AA4.1. The more differentiated subset generated only B lymphoid cells in 92% of total colonies assayed, lacked T lineage potential, and expressed Pax5. These studies have therefore defined and isolated a B lymphoid-committed progenitor population at a developmental stage corresponding to the initial expression of CD45R.     

Phenotypic and functional abnormalities of bone marrow-derived dendritic cells in systemic lupus erythematosus

Introduction  

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoreactive T and B cells, which are believed to be secondary to deficient dendritic cells (DCs). However, whether DC abnormalities occur during their development in the bone marrow (BM) or in the periphery is not known.     

Phenotypic and functional heterogeneity of EBV epitope-specific CD8+ T cells

High frequencies of EBV-specific CD8(+) T cells have been detected during acute EBV infection, yet persistent infection inevitably results. To address this issue, we characterized the phenotype and function of epitope-specific CD8(+) T cell populations from presentation with acute through latent infection. Considerable phenotypic and functional heterogeneity within, as well as between, two different epitope-specific populations was observed over time following acute infection. B7 EBV-encoded nuclear Ag (EBNA)-3A-specific CD8(+) T cells expressed only CD45RO from acute through latent EBV infection. A2 BMLF-1-specific CD8(+) T cells expressed CD45RO during acute infection and either CD45RA or CD45RO during latent EBV infection. This difference in CD45 isoform expression between the two epitope-specific populations did not translate into differences in perforin content, the ability to produce IFN-gamma, or the ability to proliferate in response to Ag in vitro. In individuals with latent EBV infection, the frequencies of A2 BMLF-1- or B7 EBNA-3A-specific CD8(+) T cells that expressed CD45RA, CD45RO, CD62 ligand, CCR7, and perforin were stable over time. However, the expression of CD62 ligand and CCR7 was significantly higher among EBNA-3A-specific CD8(+) T cells than among BMLF-1-specific CD8(+) T cells. Further work is necessary to understand how phenotypic and functional differences between EBV epitope-specific CD8(+) T cells are related to the biology of the virus and to the equilibrium between the virus and the host during persistent infection.     

Estrogen selectively promotes the differentiation of dendritic cells with characteristics of Langerhans cells

The steroid hormone estrogen regulates the differentiation, survival, or function of diverse immune cells. Previously, we found that physiological amounts of 17beta-estradiol act via estrogen receptors (ER) to promote the GM-CSF-mediated differentiation of dendritic cells (DC) from murine bone marrow progenitors in ex vivo cultures. Of the two major subsets of CD11c(+) DC that develop in these cultures, estrogen is preferentially required for the differentiation of a CD11b(int)Ly6C(-) population, although it also promotes increased numbers of a CD11b(high)Ly6C(+) population. Although both DC subsets express ERalpha, only the CD11b(high)Ly6C(+) DC express ERbeta, perhaps providing a foundation for the differential regulation of these two DC types by estrogen. The two DC populations exhibit distinct phenotypes in terms of capacity for costimulatory molecule and MHC expression, and Ag internalization, which predict functional differences. The CD11b(int)Ly6C(-) population shows the greatest increase in MHC and CD86 expression after LPS activation. Most notably, the estrogen-dependent CD11b(int)Ly6C(-) DC express langerin (CD207) and contain Birbeck granules characteristic of Langerhans cells. These data show that estrogen promotes a DC population with the unique features of epidermal Langerhans cells and suggest that differentiation of Langerhans cells in vivo will be dependent upon local estrogen levels and ER-mediated signaling events in skin.     

Langerhans cells

Epidermal Langerhans cells, a constituent of the skin immune system, have a spectrum of different functions with implications that extend far beyond the skin. They have the potential to internalize particulate agents and macromolecules, and display migratory properties that endow them with the unique capacity to journey between skin and draining lymph nodes where they encounter antigen-specific T lymphocytes. In addition, LC are considered to play a pivotal role in infectious disease such as Aids, allergy, chronic inflammatory reactions, tumor rejections or transplantation. Herein, we will review the features of Langerhans cells, emphasizing characteristics representative of their life-cycle stages that occur within the skin.     

Phenotypic and functional deficiency of natural killer cells in patients with chronic fatigue syndrome

Natural killer (NK)3 cells are large granular lymphocytes that appear to play a significant role in the host's defense against viral infection. We performed an extensive phenotypic and functional characterization of NK cells on 41 patients with the chronic fatigue syndrome (CFS), or "chronic active Epstein-Barr virus infection" syndrome, and on 23 age- and sex-matched asymptomatic control subjects in an attempt to further characterize this illness. These studies demonstrated that a majority of patients with CFS have low numbers of NKH1+T3- lymphocytes, a population that represents the great majority of NK cells in normal individuals. CFS patients had normal numbers of NKH1+T3+ lymphocytes, a population that represents a relatively small fraction of NK cells in normal individuals. When tested for cytotoxicity against a variety of different target cells, patients with CFS consistently demonstrated low levels of killing. After activation of cytolytic activity with recombinant interleukin 2, patients were able to display increased killing against K562 but most patients remained unable to lyse Epstein-Barr virus-infected B cell targets. Additional cytotoxicity experiments were carried out utilizing anti-T3 monoclonal antibody to block killing by NKH1+T3+ cells. These experiments indicated that the NK cell that appears to be responsible for much of the functional activity remaining in patients with CFS belongs to the NKH1+T3+ subset, which under normal circumstances represents only approximately 20% of the NK cell population.