Simultaneous Purification of Round and Elongated Spermatids from Testis Tissue Using a FACS-Based DNA Ploidy Assay.

Spermiogenesis is the final phase of spermatogenesis during which post-meiotic haploid round spermatids (rSpt) differentiate into elongated spermatozoa and includes several critical cell-specific processes like DNA condensation, formation of the acrosome, and production of the flagellum. Disturbances in this process will lead to complications in sperm development and subsequently cause infertility. As such, studying spermiogenesis has clinical relevance in investigating the etiology of male infertility and will improve our scientific understanding of male germ cell formation. Here, we were able to purify round spermatid and elongated spermatid fractions from a single cryopreserved human testicular tissues sample with an efficiency of 85.4% ± 4.9% and 97.6% ± 0.6%, respectively. We confirmed the cell types by morphology and immunohistochemistry for histone H4 and PNA protein expression. The purity was measured by manual counting of histone H4 positive (round) and negative (elongated) spermatids in both sorted 1 N cell fractions. This method can be applied to both human and rodent studies. Especially in studies with limited access to testicular tissue, this method provides a reliable means to simultaneously isolate these cell types with high purity. Our method allows for further investigation of germ cell development and the process of spermiogenesis in particular, as well as provides a tool to study the etiology of male infertility, including morphological and biochemical assessment of round and elongating spermatids from subfertile men. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

High IL-13 production by human neonatal T cells: neonate immune system regulator?

Neonates are highly susceptible to diseases and display biased type 2 immune responses, although no skewing to type 2 cytokines has been reported. In view of the emerging importance of IL-13 in type 2 inflammatory responses and clinical allergy, we analyzed IL-13 production by neonatal T cells. We found that, mainly CD8 T cells produced high levels of IL-13, while producing low levels of IL-4, IL-10 and IFN-gamma, upon primary and secondary stimulation. Our results point towards a possible immunoregulatory role of CD8 T cells in neonate responses. Moreover, they suggest that the abundance of IL-13 in the neonate immune system might account for the type 2 bias in neonates, providing a basis for the high disease susceptibility of newborns, for instance to allergic diseases.

Cytolytic mechanisms and expression of activation-regulating receptors on effector-type CD8+CD45RA+CD27- human T cells.

Circulating CD8+ T cells with a CD45RA+CD27- phenotype resemble cytolytic effector cells because they express various cytolytic mediators and are able to execute cytotoxicity without prior stimulation in vitro. We here demonstrate that CD8+CD45RA+CD27- T cells can use both granule exocytosis and Fas/Fas ligand pathways to induce apoptosis in target cells. The availability of these cytolytic mechanisms in circulating T cells suggests that the activity of these cells must be carefully controlled to prevent unwanted tissue damage. For this reason, we analyzed the expression of surface receptors that either enhance or inhibit T cell function. Compared with memory-type cells, effector cells were found to express normal levels of CD3epsilon and TCRzeta and relatively high levels of CD8. CTLA-4 was absent from freshly isolated effector cells, whereas a limited number of unstimulated memory cells expressed this molecule. In line with recent findings on CD8+CD28- T cells, CD45RA+CD27- T cells were unique in the abundant expression of NK cell-inhibitory receptors, both of Ig superfamily and C-type lectin classes. Binding of NK cell-inhibitory receptors to classical and nonclassical MHC class I molecules may inhibit the activation of the cytolytic machinery induced by either Ag receptor-specific or nonspecific signals in CD8+CD45RA+CD27- T cells.

In vivo HIV-1 infection of CD45RA(+)CD4(+) T cells is established primarily by syncytium-inducing variants and correlates with the rate of CD4(+) T cell decline.

Switch from non-syncytium-inducing (NSI) to syncytium-inducing (SI) HIV type 1 (HIV-1) is associated with accelerated CD4(+) T cell depletion, which might partially be explained by higher virulence of SI variants compared with NSI variants. Because NSI and SI variants use different coreceptors for entry of target cells, altered tropism might offer an explanation for increased pathogenesis associated with SI HIV-1 infection. To investigate whether SI and NSI HIV-1 variants infect different CD4(+) T cell subsets in vivo, the distribution of SI and NSI variants over CD4(+) memory (CD45RA(-)RO(+)) and naive (CD45RA(+)RO(-)) cells was studied by using limiting dilution cultures. In contrast to NSI variants that were mainly present in CD45RO(+) cells, SI variants were equally distributed over CD45RO(+) and CD45RA(+) cells. Infection of memory cells by both NSI and SI HIV-1 and infection of naive cells primarily by SI HIV-1 corresponded closely with the differential cell surface expression of CXCR4 and CCR5. The frequency of SI-infected CD45RA(+) CD4(+) T cells, but not the frequency of NSI- or SI-infected CD45RO(+) CD4(+) T cells, correlated with the rate of CD4(+) T cell depletion. Infection of naive cells by SI HIV-1 may interfere with CD4(+) T cell production and thus account for rapid CD4(+) T cell depletion.

Diversity of the T-cell receptor BV repertoire in HIV-1-infected patients reflects the biphasic CD4+ T-cell repopulation kinetics during highly active antiretroviral therapy.

OBJECTIVES: Highly active antiretroviral therapy (HAART) induces a decline in viral load and a biphasic increase in peripheral blood CD4+ T-cell counts in HIV-infected patients. To evaluate the effect of HAART on T-cell receptor (TCR) diversity of repopulating naive and memory CD4+ T cells, complementarity determining region 3 (CDR3) spectratyping was performed. DESIGN: For four patients treated with HAART, CD45RO+ (memory) and CD45RA+ (naive) CD4+ T cells were isolated from peripheral blood leukocyte samples obtained 1 week before, 1-2 months after, and 9-11 months after start of treatment. METHODS: CDR3 regions were amplified by TCR-BV-specific nested PCR from CD4+ T-cell subsets. CDR3 size distributions and single-strand conformation polymorphism profiles were compared as an indication for TCR diversity. RESULTS: Increasing blood CD4+ T-cell counts during the first 2 months of treatment coincided with increased perturbation of CDR3 patterns in CD4+ T-cell subsets, suggesting an early oligoclonal repopulation. At later timepoints, CDR3 size diversity increased when T-cell counts did not substantially decrease. Memory and naive CD4+ T cells generally showed comparable levels of perturbation. CONCLUSION: Diversity of the TCR repertoire reflected biphasic T-cell repopulation during HAART, compatible with initial redistribution and later CD4+ T-cell production. Sustained elevation of T-cell counts will in principle result in restoration of TCR diversity.

Phenotypic and functional separation of memory and effector human CD8+ T cells.

Human CD8+ memory- and effector-type T cells are poorly defined. We show here that, next to a naive compartment, two discrete primed subpopulations can be found within the circulating human CD8+ T cell subset. First, CD45RA-CD45R0(+) cells are reminiscent of memory-type T cells in that they express elevated levels of CD95 (Fas) and the integrin family members CD11a, CD18, CD29, CD49d, and CD49e, compared to naive CD8+ T cells, and are able to secrete not only interleukin (IL) 2 but also interferon gamma, tumor necrosis factor alpha, and IL-4. This subset does not exert cytolytic activity without prior in vitro stimulation but does contain virus-specific cytotoxic T lymphocyte (CTL) precursors. A second primed population is characterized by CD45RA expression with concomitant absence of expression of the costimulatory molecules CD27 and CD28. The CD8+CD45RA+CD27- population contains T cells expressing high levels of CD11a, CD11b, CD18, and CD49d, whereas CD62L (L-selectin) is not expressed. These T cells do not secrete IL-2 or -4 but can produce IFN-gamma and TNF-alpha. In accordance with this finding, cells contained within this subpopulation depend for proliferation on exogenous growth factors such as IL-2 and -15. Interestingly, CD8+CD45RA+CD27- cells parallel effector CTLs, as they abundantly express Fas-ligand mRNA, contain perforin and granzyme B, and have high cytolytic activity without in vitro prestimulation. Based on both phenotypic and functional properties, we conclude that memory- and effector-type T cells can be separated as distinct entities within the human CD8+ T cell subset.

Analysis of variation in results of flow cytometric lymphocyte immunophenotyping in a multicenter study.

Fifty-five laboratories participated in a send-out study of four peripheral blood samples comparing a standard protocol vs. local protocols for flow cytometric lymphocyte immunophenotyping. The standard protocol included centrally provided reagents, instrument setup using triple-fluorescent microbeads and a three-color, whole-blood immunostaining technique based on fluorescein isothiocyanate and phycoerythrin-labeled monoclonal antibodies, erythrocyte lysis, washing, fixation, and identification of nucleated cells by the DNA/RNA stain LDS-751. Data analysis guidelines included lymphocyte selection using CD45,CD14-assisted "backgating" on forward (FSC) and sideward (SSC) light scatter and placement of fluorescence (FL) markers on the basis of the isotype control staining. Most (i.e., 77%) of the variation in results of percentage lymphocyte subset assessments using the standard protocol was explained by laboratory, sample, background FL, and the interaction between laboratory and sample. Purity and completeness of the FSC,SSC lymphogate, background FL, flow cytometer type, and flow cytometer setup (which were either partly or entirely determined by laboratory) contributed significantly to the variation. The effect of the leukocyte differential count on the variation in absolute numbers of lymphocyte subsets was particularly large in lymphopenic samples. The use of this standard protocol vs. local protocols did not reduce the interlaboratory variation. Instrument incompatibility with the standard protocol (e.g., incompatible filter combinations for LDS-751 detection) and lack of experience of many participants with three-color flow cytometry (in particular with the use of LDS-751) may have contributed to that result. We suggest that training and experience in a universally applicable standard protocol are critical for minimization of interlaboratory variation in flow cytometric immunophenotyping.

Reduction of interlaboratory variability in flow cytometric immunophenotyping by standardization of instrument set-up and calibration, and standard list mode data analysis.

Two workshops addressed the question to which degree standardization of instrument set-up and calibration, and standard list mode data analysis would reduce interlaboratory variability of flow cytometric results on prestained peripheral blood mononuclear cells (PBMC). Standard instrument set-up included uniform positioning of the "windows of analysis" for the forward and sideward light scatter and fluorescence (FL) 1 (i.e., fluorescein isothiocyanate [FITC]) and 2 (i.e., phycoerythrin [PE]) parameters. Reference standards and PBMC, double-stained with FITC- and PE-conjugated monoclonal antibodies covering a wide range of FL intensities and coexpression patterns, were sent out to 25 laboratories in Workshop 1 and to 35 laboratories in Workshop 2 with the following requests: a) to set up instruments according to local and standard protocols, b) to acquire list mode data on the PBMC with both instrument settings, and c) to analyze both datasets according to local protocols. Standard analysis of the list mode data acquired with uniform instrument settings was performed centrally using so-called "latent class model" software (Van Putten et al., Cytometry 14:86-96, 1993). This software provides an automated, "no-gating" analytical method of lymphocyte immunophenotypes and employs fixed FL marker settings as defined prior to each analytical run. In Workshop 1, these markers were set in identical histogram channels for all instruments based on results obtained with a reference instrument. Standard analysis of list mode data acquired after uniform instrument set-up led only to a 13% reduction of interlaboratory variability of results as compared to data analysis using local protocols. The standard protocol for instrument set-up led to uniform positioning of relatively strong FL signals but variable positioning of unstained cells on the FL histogram scales. Hence, standard FL marker settings were inappropriate for some instruments. Therefore, instrument responses to FITC and PE signals in Workshop 2 were calibrated using microbeads labeled with FITC or PE in a range of predefined FL intensities expressed in MESF units (molecules of equivalent soluble fluorochrome). That approach allowed the positioning of the FL markers for the standard analysis on the basis of identical FL1 and FL2 intensities, expressed in MESF units, for all instruments. Standard analysis of list mode data acquired after uniform instrument set-up and calibrated FL marker settings led to a 43% reduction of interlaboratory variability as compared to data analysis to local protocols. We conclude that standard list mode data analysis using fixed FL marker settings reduces the interlaboratory variability of flow cytometric results on prestained PBMC, provided that the instruments have been set up in a uniform way and that FL markers have been standardized on the basis of calibration of each instrument's response to the corresponding FL signals.

Antigen-presenting cell-derived signals determine expression levels of CD70 on primed T cells.

Interaction between CD27 and its ligand CD70 provides a second signal for T-cell proliferation and tumour necrosis factor-alpha (TNF-alpha) production. Whereas CD27 is broadly expressed during T-cell development, expression of CD70 in vivo is restricted. To determine when CD27 CD70 interactions can occur in immune reactions, we here analysed the regulation of CD70 expression on activated T cells. Mitogenic stimulation of purified T cells with either immobilized CD3 monoclonal antibody (mAb) or a combination of CD2 mAb induces only low levels of CD70 membrane expression. Markedly expression of the CD27-ligand is strongly enhanced by antigen-presenting cells (APC) and APC-associated signals such as interleukin-1 alpha (IL-1 alpha). IL-12, TNF-alpha and CD28-ligation. In contrast, T-cell derived cytokines, such as IL-4, counteract CD70 up-regulation on activated T cells. Analysis of the small subset of circulating CD70+ T cells revealed that these cells have a primed phenotype as they express CD45RO and HLA-DR antigens and are in high frequency able to secrete interferon-gamma (IFN-gamma). We conclude that T-T interactions involving CD27 and CD70 are likely to occur relatively early in immune reactions, after productive T-cell priming by APC and that expression of CD70 on circulating T cells is a reflection of recent priming by antigen.

Phenotype and function of human B cells expressing CD70 (CD27 ligand).

CD70, the cellular ligand of the tumor necrosis factor receptor family member CD27, can be found on a limited number of germinal center (GC) B cells in some tonsils, on scattered lymphocytes residing in secondary lymphoid organs, and on a fraction of the circulating B cell population. Due to the restricted expression of CD70 in vivo, we analyzed signals that determine CD70 expression levels and characterized the phenotype and function of CD70+ B cells. Expression of CD70 on B cells activated in vitro was found to be dependent on the continuous presence of a B cell antigen receptor cross-linking agent, and induced or potentiated by CD40 ligation but was down-modulated by the Th2 cytokines interleukin (IL)-4 and IL-13. Both in peripheral blood and tonsil cell suspensions, CD70+ B cell subpopulations were found to be enriched for CD27- and IgG-expressing cells, but contained less IgD+ B cells. Additional analysis of markers which define specific differentiation stages (Bm1-5) of mature B cells within human tonsils did not place CD70-expressing B cells in one of these subsets. Functional experiments revealed that whereas both CD70- and CD70+ B cells can secrete immunoglobulin after activation with a combination of Staphylococcus aureus Cowan strain I and IL-2, only CD70+ B cells can produce large quantities of antibodies when stimulated in a T cell-dependent fashion. Our combined data imply that CD70 is a marker for mature B cells which have recently been primed by antigen in vivo.

Heterogeneity of the human CD4+ T-cell population: two distinct CD4+ T-cell subsets characterized by coexpression of CD45RA and CD45RO isoforms.

Activation of unprimed CD4+CD45RA+/RO- T cells results in a gradual loss of CD45RA expression concomitant with the acquisition of CD45RO. It has been suggested that this conversion occurs in vivo through a CD45RAbright/RObright stage. Next to this small CD45RAbright/RObright subset (Dbright), a larger subpopulation that expresses both RA and RO isoforms at low levels (Ddull) can be found in the circulating CD4+ T-cell population of all donors. The properties of the latter population are largely undefined. Here, we show that Ddull cells have an intermediate phenotype for antigens such as CD31, CD621, CD58, and CD95 that are differentially expressed on unprimed versus primed T cells. In addition, they are able to provide help for B-cell differentiation and contain substantial numbers of tetanus toxoid (TT)-specific precursor cells. Remarkably, both intracellular cytokine staining and analysis of T-cell clones showed that Ddull cells and CD45RO+ T cells produce comparable high amounts of both interferon (IFN)-gamma and interleukin (IL)-4, which clearly distinguishes them from CD45RA+ and Dbright T cells. Finally, prolonged culture of sorted Ddull cells in a mixture of IL-2, IL-6, and tumor necrosis factor (TNF)-alpha showed that about half of the population retained the Ddull phenotype. Part of the cells upregulated the CD45RA isoform, whereas only a minority switched to single CD45RO expression. Our findings indicate that the Ddull population contains primed T cells, some of which may reacquire an "unprimed" phenotype in the absence of antigenic stimulation.

Single cell analysis of IL-4 and IFN-gamma production by T cells from HIV-infected individuals: decreased IFN-gamma in the presence of preserved IL-4 production.

The specific in vitro disturbance of capacities ascribed to Th1 cells in HIV-infected individuals suggests a switch from Th1 to Th2 lymphokine secretion. Indeed, when T cell clones are generated from HIV-infected individuals compared with controls, an increased percentage of Th0 clones is present upon HIV infection. We studied cytokine production in the supernatant of in vitro activated PBMC from a large group of HIV-infected patients at various stages of infection. IL-2, IFN-gamma, IL-4, IL-5, and IL-10 production all were decreased significantly, which does not support a switch to Th2 lymphokine secretion and is possibly due to the generalized impaired response of T cells from HIV-infected individuals to activation signals in vitro. Therefore, we investigated the capacity of single cells to produce a certain cytokine. Intracellular staining of IL-4- and IFN-gamma-producing cells revealed that T cells from HIV-infected individuals contained decreased numbers of IFN-gamma-producing cells, in the presence of normal percentages of cells with the capacity to produce IL-4. This resulted in significantly decreased IFN-gamma/IL-4 ratios in both CD4+ and CD8+ T cells. Thus, in agreement with previous findings in T cell clones, we conclude, from cytokine production upon stimulation of T cells in vitro, that there is a change in the cytokine balance to the Th2 side in HIV infection due to decreased Th1 and preserved Th2 cytokine production.

Alteration of B-cell antigen receptor signaling by CD19 co-ligation. A study with bispecific antibodies.

The activation of B-cell antigen receptor-associated protein tyrosine kinases is an early and crucial event in B-cell signaling. Apart from the B-cell antigen receptor (BCR), the B-cell-specific transmembrane glycoprotein CD19 has also been shown to directly activate intracellular signaling cascades. In addition, because CD19 and the BCR are associated on the surface of activated B-cells, it has been proposed that close approximation between these two entities is crucial for optimal B-cell triggering. To test this hypothesis, bispecific antibodies were generated that bind membrane IgM and CD19 simultaneously. Although CD19 bispecific antibodies strongly induced tyrosine phosphorylation, they were, in contrast to muF(ab)2 fragments, unable to induce a proliferative response. Detailed analysis of the early signaling events showed that compared with muF(ab)2 fragments CD19 bispecific antibodies potently raised the intracellular [Ca2+], which was correlated with an efficient tyrosine phosphorylation of syk. Strikingly, the assembly of Grb2 complexes that may couple the BCR to p21(ras) was clearly altered by the CD19 bispecific antibody. In addition to the reported Shc and 145-kDa phosphoproteins, a prominent 90-95-kDa phosphoprotein resembling CD19 was detected in the Grb2 complexes. Thus, studies with CD19 bispecific antibodies show that CD19 co-ligation both quantitatively and qualitatively alters BCR signaling.

Isolation and culture of human bone marrow endothelial cells.

Bone marrow endothelial cells are likely to play an important role in the homing of hematopoietic progenitor cells. In view of analyzing the interactions between endothelial cells and hematopoietic progenitor cells, we studied several methods of isolating endothelial cells from human bone marrow, including fluorescence activated cell sorting (FACS) and separation by immunomagnetic beads. FACS sorting gave the best results as contamination with other cells did not occur. After density-gradient centrifugation of bone marrow aspirates, the mononuclear cell (MNC) fraction was depleted for T cells, B cells, and myeloid cells by immunomagnetic separation. Further enrichment of endothelial cells was achieved by FACS sorting using BNH9 or S-Endo1 monoclonal antibodies (MAbs). These MAbs, in contrast to several other endothelial-cell reactive MAbs, were found to react highly specifically with sinus endothelial cells as tested by immunohistochemistry on bone marrow tissue sections and cell culture preparations and by double-colored FACS analysis on bone marrow MNCs (BMMNC). Sorted cells, which formed 0.05% of the MNC fraction, showed strong intracytoplasmic von Willebrand factor positivity. Ultrastructural analysis revealed cells with endothelial characteristics. Cells were cultured in fibronectin-coated, 24-well culture plates in endothelial-cell culture medium or long-term bone marrow culture medium. After 1 to 3 weeks of culture, a monolayer of spindle-shaped cells developed expressing endothelial cell antigens. Cells could be kept in culture for 4 to 6 weeks. In conclusion, the method described provides highly purified preparations of human bone marrow endothelium that may permit in vitro adhesion experiments with normal and leukemic hematopoietic progenitor cells.

Heterogeneity of the circulating human CD4+ T cell population. Further evidence that the CD4+CD45RA-CD27- T cell subset contains specialized primed T cells.

CD27, a member of the TNFR family, is expressed on most but not all peripheral blood CD4+ T cells. The small fraction of CD4+ T cells with a CD27- phenotype exclusively reside within the CD45RA-CD45RO+ subset. We previously provided evidence that CD27- cells are functionally differentiated cells that have lost CD27 expression as a result of persistent antigenic stimulation. We here show that compared with CD4+CD45RA-CD27+ cells, CD4+CD45RA-CD27- lymphocytes have a high expression of the beta 1 integrins VLA-4 and -5 and of the beta 2 integrin CD11b. Molecules implicated in homing of T cells to peripheral lymphnodes like CD31 and CD62L (L-selectin) are poorly expressed on CD27- cells, whereas receptors involved in organ-specific homing, e.g., cutaneous lymphocyte Ag and HML-1 (alpha E beta 7), are present on CD27- rather than CD27+ T lymphocytes. CD27+ and CD27- cells do not differ notably in the expression of activation molecules such as CD25, CD38, and CD70 (CD27 ligand) but CD7 is markedly absent on approximately half of the CD27- cells. Analysis of mutations in the HPRT gene, as measurement for the amount of cell divisions that have occurred in particular T cell populations in vivo, showed that CD45R0+ cells have a 2 to 5 times higher mutant frequency than CD45RA+ cells, whereas CD45R0+CD27- cells do not differ in this respect from CD45R0+CD27+ cells. In line with this latter finding, cells in G2M phase can only be found in the transitional, CD45RAbrightCD45R0bright subset but not in CD45R0+, CD45RA-, or CD27- cells. Our results imply that the CD27- population contains tissue-specific, specialized "primed" T cells that evolve in vivo independently from extensive cellular division.

Productive HIV-1 infection of macrophages restricted to the cell fraction with proliferative capacity.

Retroviruses establish productive infection only in proliferating cells. Macrophages are often considered to be non-proliferating in vitro yet are susceptible to HIV-1 infection. This has led to the conclusion that HIV-1 can establish infection independent of host cell proliferation. We here report that a small proportion of macrophages does have proliferative capacity. A comparable small fraction of monocyte derived macrophages (MDM) supported productive HIV-1 infection as demonstrated in limiting dilution culture. Fluorescence activated cell sorting on the basis of incorporation of BrdUrd, a thymidine analog, and subsequent PCR analysis revealed the presence of proviral DNA only in the BrdUrd positive cell fraction with DNA synthesizing activity. To identify which phase of cell cycle is required for establishment of productive infection, growth arrest in G1 or G1/S phase prior to inoculation was performed. gamma-Irradiation, which arrests primary cells in G1, prevented both cell proliferation and establishment of productive infection in MDM. Treatment of MDM with aphidicolin, a specific inhibitor of DNA polymerase alpha and delta which arrests cells in G1/S phase of the cell cycle, also inhibited DNA synthesis but did not prevent establishment of productive infection which is completely analogous to observations in T cells. Our data thus indicate that not cell division itself but cellular conditions that coincide with cell proliferation are apparently indispensable for establishment of productive infection.

Quantitative analysis of CD4+ T cell function in the course of human immunodeficiency virus infection. Gradual decline of both naive and memory alloreactive T cells.

Early in human immunodeficiency virus (HIV) infection CD4+ and CD8+ T cells are qualitatively affected. Loss of responses to recall antigen precedes impaired responses to allogeneic MHC and mitogens. The selective quantitative loss of memory T cells in early infection, only partially explains the observed defects. We investigated whether functional loss of T cells is preferentially observed for memory T cells or whether both naive and memory T cell subsets are affected in the course of HIV infection. We studied the proliferative response of CD4+ T cells from HIV-infected individuals to alloantigens, to which normally both naive and memory T cells respond, by limiting dilution analysis. The decreased proliferative response to alloantigens in HIV-infected individuals was associated with a decreased precursor frequency of alloreactive cells. The frequency was decreased in both the CD45RA+ (naive) and the CD45RO+ (memory) subset of CD4+ T cells. Analysis of four individuals in the course of HIV infection revealed similar kinetics of the decline in function in both subsets. Although initially T cell defects may be accounted for by the selective quantitative loss of memory cells, in later stages of HIV infection the function of both CD45RA+ and CD45RO+ cells is affected.

Immunophenotyping of eosinophils recovered from blood and BAL of allergic asthmatics.

Studies of bronchoalveolar lavage (BAL) fluid from patients with allergic asthma have demonstrated active migration of eosinophils into the bronchial lumen after allergen challenge. The mechanisms mediating this eosinophil infiltration and cell activation are largely unexplained. The expression of several cell-surface molecules was measured on eosinophils derived from blood and BAL fluid 4 h after an allergen-induced early asthmatic reaction in order to find indications for a role of these molecules during extravasation to and activation in the bronchial compartment. Nine patients with allergic asthma participated in the study. An eosinophil-specific, high-depolarization signal enabled us to measure expression on eosinophils in a fluorescence activated cell sorter (FACS) analysis without isolation of these cells. Eosinophils recovered from BAL showed a different phenotype than blood eosinophils; upregulation of CR-3, p150/95, CD67, and CD63, and downregulation of L-selectin indicate that the cells are activated in terms of degranulation. Up-regulation of intercellular adhesion molecule-1 (ICAM-1), LFA-3, and human leukocyte antigen II (HLA-II) might enable cell-cell contact between T-lymphocytes and eosinophils, probably leading to immunomodulation and cell activation. The finding that eosinophils in BAL are activated and can interact with T cells is further evidence for the proinflammatory role of these cells in allergic asthma.

Interleukin-6 is a survival factor for committed myeloid progenitor cells.

In this study, we investigated the effect of recombinant human interleukin-6 (IL-6) on colony-forming cells for granulocytes and macrophages (CFU-GM) cultured in suspension. IL-6 when used alone did not induce proliferation of highly purified CD34+ human hematopoietic progenitors. Moreover, no influence of IL-6 was observed on the proliferation induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) or granulocyte (G)-CSF. However, a marked survival enhancement (GM-CSF 228 +/- 42%, p < 0.01, and G-CSF 137 +/- 9%, p < 0.05) was observed when CD34+ cells were preincubated with IL-6 for 6 days. This survival effect became even more pronounced under serum-poor conditions (GM-CSF 380 +/- 80%, p < 0.01, and G-CSF 180 +/- 20%, p < 0.01) and could also be demonstrated at the single cell level in a colony-forming assay. By analysis of subpopulations of CD34+ bone marrow (BM) cells selected on the basis of CD45RO expression, the observed IL-6-mediated survival effect was found to be restricted to the CFU-GM containing CD45RO- subset. Our data show that IL-6 is a survival factor for CFU-GM.

Down modulation of L-Selectin expression on eosinophils recovered from bronchoalveolar lavage fluid after allergen provocation.

In allergic asthma eosinophils infiltrate into the lung after allergen challenge. The mechanism of this cellular infiltration is not fully understood. L-Selectin is involved in leucocyte-endothelial cell recognition and participates in homing of leucocytes into sites of inflammation. To find indications for a role of L-Selectin in the migration of eosinophils to the bronchoalveolar space we measured L-Selectin expression on eosinophils in peripheral blood and bronchoalveolar lavage fluid (BAL) 4 hr after the early allergic reaction after allergen challenge. Nine patients with allergic asthma participated in the study. An eosinophil specific high depolarization signal enabled us to measure L-Selectin expression on eosinophils in a FACS analysis without isolation of these cells. Eosinophils recovered from BAL showed a strong decrease of L-Selectin expression compared to blood eosinophils. This decrease in L-Selectin expression can be induced in vitro by activation of eosinophils with PMA or FMLP whereas priming of eosinophils during several hours with GM-CSF did not influence L-Selectin expression. Our results are a first indication that L-Selectin may play a role during homing of eosinophils in the lung in asthma after allergen challenge. Moreover, the low expression of L-Selectin on eosinophils in the lung is a further indication that these cells exhibit an activated phenotype.