Myeloid blasts are the mouse bone marrow cells prone to differentiate into osteoclasts

Cells of the myeloid lineage at various stages of maturity can differentiate into multinucleated osteoclasts. Yet, it is unclear which developmental stages of this lineage are more prone to become osteoclasts than others. We investigated the osteoclastogenic potential of three successive stages of myeloid development isolated from mouse bone marrow. Early blasts (CD31hi/Ly-6C-), myeloid blasts (CD31+/Ly-6C+), and monocytes (CD31-/Ly-6Chi), as well as unfractionated marrow cells, were cultured in the presence of M-CSF and receptor activator of NF-B ligand (RANKL), and the differentiation toward multinucleated cells and their capacity to resorb bone was assessed. Myeloid blasts developed rapidly into multinucleated cells; in only 4 days, maximal numbers were reached, whereas the other fractions required 8 days to reach maximal numbers. Bone resorption was observed after 6 (myeloid blasts and monocyte-derived osteoclasts) and 8 (early blast-derived osteoclasts) days. This difference in kinetics in osteoclast-forming capacity was confirmed by the analysis of osteoclast-related genes. In addition, the myeloid blast fraction proved to be most sensitive to M-CSF and RANKL, as assessed with a colony-forming assay. Our results show that osteoclasts can develop from all stages of myeloid differentiation, but myeloid blasts are equipped to do so within a short period of time

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 depletio

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

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 stimulatio

Properties of murine (CD8+)CD27- T cells.

In humans, loss of CD27 expression is associated with the stable acquisition of effector functions by CD8+ T cells. We found that murine (CD8+)CD27- T cells were confined to the primed CD62L(dull/-)CD44(bright)CCR7- T cell population. (CD8+)CD27- T cells were absent from lymph nodes but could be found in blood, spleen and in non-lymphoid organs such as lung and liver. Late after primary influenza virus infection, low percentages of antigen-specific CD27- cells emerged in the lung and spleen. After recovery from secondary influenza virus infection, high percentages of influenza-specific CD27- T cells were found in the lung and the loss of CD27 on lung CD8+ T cells coincided with high granzyme B expression. After murine cytomegalovirus infection, loss of CD27 expression on virus-specific CD8+ T cell populations was sustained and especially marked in liver and lung. We suggest that in mice, CD27 is lost from CD8+ T cells only after repetitive antigenic stimulation. Moreover, the high expression of both granzyme B and perforin in the CD27- T cells suggests that the lack of CD27 on murine CD8+ T cells can be used to identify memory T cells with expression of cytotoxic effector molecules