Novel repair action of vitamin C upon in vivo oxidative DNA damage

The Nef protein of HIV-1 binds to and induces apoptotic cytolysis of uninfected but activated human peripheral blood mononuclear cells (PBMC) and various cell line cells derived from CD4+ T, CD8+ T and B lymphocytes, macrophages, and neutrophils. The Nef-induced apoptosis also occurs with blood cells not expressing CD95 (Fas). The Nef-induced apoptosis as well as Fas-mediated apoptosis was inhibited by acetyl-Try-Val-Ala-Asp-CHO, an IL-1beta converting enzyme (ICE) inhibitor. On the other hand, serine/threonine protein kinase (PK) inhibitors, H-7, fasudil hydrochloride and M3, inhibited the Nef-induced apoptosis, and not the Fas-mediated one, without affecting the cell-binding activity of Nef and Nef-binding capacity of the activated cells. Preincubation of the cells with the drugs before being bound by Nef was required for the inhibition of apoptosis. These results suggest that the PK inhibitors specifically act on a cellular protein involved in the upper stream of signal transduction pathway of the Nef-induced apoptosis, which is different from the Fas-mediated pathway but meets it upstream of ICE. In addition, the drugs suppressed the cellular activation-associated cell surface expression of a putative Nef-binding protein in PBMC, although they had no influence on its expression in cell line cells. These findings suggest the feasibility of clinical use of the PK inhibitors to prevent the development of AIDS by inhibiting the Nef-induced apoptosis of uninfected blood cells.     

Mobilization and homing of peripheral blood progenitors is related to reversible downregulation of alpha4 beta1 integrin expression and function

Despite the wide use of mobilized peripheral blood (PB) progenitor cells (PBPC) for clinical transplantation the mechanism(s) underlying their mobilization and subsequent engraftment are still unknown. We compared the adhesive phenotype of CD34(+) colony-forming cells (CFC) in bone marrow (BM) and PB of normal donors before and after administration of granulocyte colony-stimulating factor (G-CSF) for 5 d. G-CSF-mobilized PB CFC cells adhered significantly less to BM stroma, fibronectin, and to the alpha4 beta1 binding fibronectin peptide, CS1, because of decreased expression of the alpha4 integrin. Since incubation of BM CD34(+) cells for 4 d with G-CSF at concentrations found in serum of G-CSF- treated individuals did not affect alpha4-dependent adhesion, G-CSF may not be directly responsible for the decreased alpha4-mediated adhesion of PB CFC. Culture of G-CSF-mobilized PB CD34(+) cells with cytokines at concentrations found in BM stromal cultures upregulated alpha4 expression and restored adhesion of mobilized PB CFC to stroma, fibronectin, and CS1. Adhesion of cultured, mobilized PB CFC to stroma and CS1 could not be further upregulated by the beta1 activating antibody, 8A2. This indicates acquisition of a maximally activated alpha4 beta1 integrin once PB CFC have been removed from the in vivo mobilizing milieu. Thus, decreased alpha4 expression on CD34(+) CFC in PB may be responsible for the aberrant circulation of mobilized PB CD34(+) cells. Reexpression of a maximally activated alpha4 beta1 integrin on mobilized PB CFC removed from the mobilizing in vivo milieu may contribute to the early engraftment of mobilized PBPC.     

Isolation and characterization of a neuropathogenic simian immunodeficiency virus derived from a sooty mangabey

Transfusion of blood from a simian immunodeficiency virus (SIV)- and simian T-cell lymphotropic virus-infected sooty mangabey (designated FGb) to rhesus and pig-tailed macaques resulted in the development of neurologic disease in addition to AIDS. To investigate the role of SIV in neurologic disease, virus was isolated from a lymph node of a pig-tailed macaque (designated PGm) and the cerebrospinal fluid of a rhesus macaque (designated ROn2) and passaged to additional macaques. SIV-related neuropathogenic effects were observed in 100% of the pig-tailed macaques inoculated with either virus. Lesions in these animals included extensive formation of SIV RNA-positive giant cells in the brain parenchyma and meninges. Based upon morphology, the majority of infected cells in both lymphoid and brain tissue appeared to be of macrophage lineage. The virus isolates replicated very well in pig-tailed and rhesus macaque peripheral blood mononuclear cells (PBMC) with rapid kinetics. Differential replicative abilities were observed in both PBMC and macrophage populations, with viruses growing to higher titers in pig-tailed macaque cells than in rhesus macaque cells. An infectious molecular clone of virus derived from the isolate from macaque PGm (PGm5.3) was generated and was shown to have in vitro replication characteristics similar to those of the uncloned virus stock. While molecular analyses of this virus revealed its similarity to SIV isolates from sooty mangabeys, significant amino acid differences in Env and Nef were observed. This virus should provide an excellent system for investigating the mechanism of lentivirus-induced neurologic disease.     

Generation and functional characterization of human dendritic cells derived from CD34 cells mobilized into peripheral blood: comparison with bone marrow CD34+ cells

Dendritic cells (DCs) are the most powerful professional antigen-presenting cells (APC), specializing in capturing antigens and stimulating T-cell-dependent immunity. In this study we report the generation and characterization of functional DCs derived from both steady-state bone marrow (BM) and circulating haemopoietic CD34+ cells from 14 individuals undergoing granulocyte colony-stimulating factor (G-CSF) treatment for peripheral blood stem cells (PBSC) mobilization and transplantation. Clonogenic assays in methylcellulose showed an increased frequency and proliferation of colony-forming unit-dendritic cells (CFU-DC) in circulating CD34+ cells, compared to that of BM CD34+ precursors in response to GM-CSF and TNF-alpha with or without SCF and FLT-3L. Moreover, peripheral blood (PB) CD34+ cells generated a significantly higher number of fully functional DCs, as determined by conventional mixed lymphocyte reactions (MLR), than their BM counterparts upon different culture conditions. DCs derived from mobilized stem cells were also capable of processing and presenting soluble antigens to autologous T cells for both primary and secondary immune response. Replacement of the early-acting growth factors SCF and FLT-3L with IL-4 at day 7 of culture of PB CD34+ cells enhanced both the percentage of total CD1a+ cells and CD1a+ CD14- cells and the yield of DCs after 14 d of incubation. In addition, the alloreactivity of IL-4-stimulated DCs was significantly higher than those generated in the absence of IL-4. Furthermore, autologous serum collected during G-CSF treatment was more efficient than fetal calf serum (FCS) or two different serum-free media for large-scale production of DCs. Thus, our comparative studies indicate that G-CSF mobilizes CD34+ DC precursors into PB and circulating CD34+ cells represent the optimal source for the massive generation of DCs. The sequential use of early-acting and intermediatelate-acting colony-stimulating factors (CSFs) as well as the use of autologous serum greatly enhanced the growth of DCs. These data may provide new insights for manipulating immunocompetent cells for cancer therapy.     

Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20

Murine monoclonal antibody 2B8 specifically recognizes the CD20 phosphoprotein expressed on the surface of normal B lymphocytes and B-cell lymphomas. The light- and heavy-chain variable regions of 2B8 were cloned, after amplification by the polymerase chain reaction, into a cDNA expression vector that contained human IgG1 heavy chain and human kappa-light chain constant regions. High-level expression of chimeric-2B8 antibody (C2B8) was obtained in Chinese hamster ovary cells. Purified C2B8 exhibited antigen binding affinity and human-tissue reactivity similar to the native murine antibody. In vitro studies showed the ability of C2B8 to bind human C1q, mediate complement-dependent cell lysis of human B-lymphoid cell lines, and lyse human target cells through antibody-dependent cellular cytotoxicity. Infusion of macaque cynomolgus monkeys with doses ranging from 1.6 mg/kg to 6.4 mg/kg resulted in greater than 98% depletion of peripheral blood (PB) B cells and 40% to 70% depletion of lymph node B cells. Recovery of PB B cells usually started at 2 weeks after treatment and required 60 to greater than 90 days to reach normal levels. As much as 95% depletion of B cells in peripheral lymph nodes and bone marrow was observed following weekly injections of 16.8 mg/kg antibody. No toxicity was observed in any of the animals. These results offer the possibility of using an "immunologically active" chimeric anti-CD20 antibody as an alternative approach in the treatment of B-cell lymphoma.     

Binding of HIV-1 Nef to a novel thioesterase enzyme correlates with Nef-mediated CD4 down-regulation

Nef is a 27-kDa myristoylated protein conserved in primate lentiviruses. In vivo, simian immunodeficiency virus Nef is required in macaques to produce a high viral load and full pathological effects. Nef has at least three major effects in vitro, induction of CD4 down-regulation, alteration of T cell activation pathways, and enhancement of viral infectivity. We have used the yeast two-hybrid system to identify cellular proteins that interact with HIV-1Lai Nef and could mediate Nef function. A human cDNA was isolated that encodes a new type of thioesterase, an enzyme that cleaves thioester bonds. This novel thioesterase is unlike the animal types I and II thioesterases previously cloned but is homologous to the Escherichia coli thioesterase II. Nef and this thioesterase interact in vitro and are co-immunoprecipitated by anti-Nef antibodies in CEM cells expressing Nef. Nef alleles from human immunodeficiency virus-1 (HIV-1) isolates unable to down-regulate CD4 do not react or react poorly with thioesterase. An HIV-1 NefLai mutant selected for its lack of interaction with thioesterase was also unable to down-regulate CD4 cell-surface expression. These observations suggest that this human thioesterase is a cellular mediator of Nef-induced CD4 down-regulation.     

Selective inhibition of hepatic collagen accumulation in experimental liver fibrosis in rats by a new prolyl 4-hydroxylase inhibitor

In order to detect and characterize allergen-specific T cells in the airways of atopic asthmatics, we measured proliferation and cytokine production by bronchoalveolar lavage (BAL) T cells isolated from Dermatophagoides pteronyssinus (Der p)-sensitive asthmatics and nonatopic control subjects, and compared the results with those generated using peripheral blood (PB) T cells. BAL and PB mononuclear cells were collected 24 h after segmental allergen challenge by fibreoptic bronchoscopy and venepuncture, respectively. T cells purified from BAL and PB were stimulated with autologous, irradiated antigen-presenting cells and D. pteronyssinus extract or a control, nonallergen antigen (M. tuberculosis purified protein derivative [PPD]). IL-5 and IFN-gamma concentrations were measured in culture supernatants by ELISA, and T-cell proliferation by 3H-thymidine uptake. D. pteronyssinus-induced proliferation of T cells derived from both BAL and PB was elevated in asthmatics when compared with control subjects (p < 0.05), whereas PPD-induced proliferation was equivalent in both compartments. In the asthmatics, D. pteronyssinus-induced proliferative responses of equivalent numbers of BAL and PB T cells obtained after allergen challenge were statistically equivalent. Nevertheless, BAL T cells stimulated with D. pteronyssinus produced significantly greater amounts of IL-5 than did PB T cells (p < 0.05). Allergen-induced proliferation and IL-5 production by BAL T cells in the asthmatics after segmental allergen challenge correlated with the percentages of eosinophils in the BAL fluid (p < 0.01). Further, BAL T cells from asthmatic patients produced significantly higher amounts of IL-5 than did the same number of cells from nonatopic control subjects (p < 0.05). We conclude that, in D. pteronyssinus-sensitive asthmatics, allergen-specific T cells can be detected in the bronchial lumen after allergen challenge and that allergen-induced proliferation and IL-5 production by these cells correlates with local eosinophil influx. Although bronchial luminal T cells show an equivalent proliferative response to allergen stimulation as compared with PB T cells, they do produce more IL-5, consistent with the hypothesis that local differentiation or priming of these cells within the bronchial mucosal environment results in upregulation of allergen-induced IL-5 secretion.     

Impairment of excitatory amino acid transport in astroglial cells infected with the human immunodeficiency virus type 1

Perturbation of astrocyte functions by HIV-1 infection may contribute to the pathogenesis of AIDS dementia complex (ADC). The present study investigated the possibility that astroglial transport of glutamate and aspartate, the major excitatory amino acids (EAAs) in the mammalian central nervous system (CNS), is altered by HIV-1 infection. Human U251 glioma cells were infected with the brain isolate SF162 of HIV-1. HIV-1 persisted in glial cells over several months. This nonproductive infection of glial cells was characterized by persistent expression of Nef over the time of the infection, and the transient presence of structural viral proteins, including the viral transmembrane glycoprotein gp41, which was detected during the initial 2 weeks following HIV-1 infection. The presence of gp41 in acutely HIV-1-infected glial cells coincided with a 36% decrease in D-[3H]aspartate uptake, owing to a reduction in the maximal transport capacity (vmax) for D-aspartate. The expression of typical astrocytic glutamate transporters EAAT1 and EAAT2 in U251 glioma cells was not altered by HIV-1 infection. To determine whether viral protein gp120, gp41, or Nef was involved in the impairment of EAA transport in acutely HIV-1-infected glial cells, effects of lentiviral lytic peptide type 1 (LLP-1) (corresponding to the carboxy terminus of gp41), recombinant SF2 gp120, and recombinant LAI Nef on D-[3H]aspartate uptake and the release of glutamate in glial cells were investigated. Only LLP-1 reduced D-[3H]aspartate uptake and facilitated the release of glutamate from glial cells in a concentration-dependent manner. These results suggest that the carboxy terminus of gp41 impairs EAA transport in glial cells, which may contribute to excitotoxic damage to neurons in HIV-1 infection of the CNS.     

HIV-1 Nef protein downregulation of CD4 surface expression: relevance of the lck binding domain of CD4

HIV Nef protein downregulates the surface expression of CD4 on T-cells but its mechanism of activity is unknown. We have analyzed the relevance of different portions of the CD4 molecule with respect to the activity of Nef. Upon transfection of Jurkat T-cells that express Nef or do not express Nef with constructs that include different domains of the CD4 molecule, we demonstrated that Nef downregulation of CD4 requires the first 30 amino acids of the CD4 cytoplasmic tail and that the lck-binding domain of CD4 was at least partially responsible for this effect. Furthermore, upon transfection of U-937 cells with an lck-expressing plasmid we showed that CD4 downregulation by Nef is significantly more efficient when lck is present. Finally, by measuring the rate of CD4 endocytosis by a novel flow cytometric method we showed that the effect of Nef on CD4 surface expression resulted from accelerated CD4 internalization.     

Expression of Na(+)-H+ exchanger isoforms NHE1 and NHE3 in kidney and blood cells of rabbit and rat

Increased peripheral blood cell Na-H exchange (NHE) and erythrocyte Na-Li countertransport activity have been reported in hypertension and diabetic nephropathy and correlated with increased activity of the renal brush border Na-H exchanger. A relationship between cation exchange activities of blood cells and renal brush border membranes might exist if both were mediated by the same NHE isoform. We generated isoform-specific antibodies to compare the expression of NHE1 and NHE3 in peripheral blood cell membranes and renal cortical membrane vesicles. An NHE1-specific monoclonal antibody reacted with a 199- to 110-kD protein in basolateral membrane fractions isolated from rabbit and rat kidney. NHE1 protein expression was also detected in erythrocytes, platelets, and lymphocytes isolated from rabbit and rat. Two polyclonal antisera generated against nonoverlapping portions of NHE3 reacted with proteins of 82 and 85 kD in brush border membrane fractions isolated from rabbit and rat kidney, respectively, but failed to detect NHE3 expression in blood cells. These data do not support the hypothesis that Na-H exchanger of Na-Li countertransport in blood cells takes place via the renal brush border membrane NHE isoform, namely NHE3.     

A prospective study of trephined bone grafts of the tibial shaft and iliac crest

BACKGROUND: Our purpose was to analyze the expression of Fas antigen on CD4+ lymphocytes in the aqueous humor (AH) and cerebrospinal fluid (CSF) of patients with Vogt-Koyanagi-Harada disease (VKH). METHODS: Using three-color flow cytometry, we assessed T-lymphocyte subsets stained with fluorescence-conjugated anti-CD3, CD4, CD8, CD29, CD45RA, CD45RO, HLA-DR, and Fas monoclonal antibodies in AH, CSF and peripheral blood (PB) from 8 patients with active VKH. RESULTS: CD3+ T cells constituted the majority of lymphocytes in AH and CSF, in contrast to with PB. The percentages of CD4+ lymphocytes in uveitic AH and CSF were significantly higher than that in PB (P < 0.01). Activated CD4+ and CD8+ cells were significantly more frequent in AH than in CSF and PB (P < 0.01). Although the percentages of CD45RA+ cells within CD4+ cells in AH and CSF were extremely low compared with those in PB, the proportions of CD29+ and CD45RO+ (memory) cells within CD4+ were much higher than those in PB (P < 0.01). Fas antigen was also highly expressed on such CD4+ cells in AH, as in other uveitis patients and on such cells in CSF. Moreover, the percentages of Fas+ and memory cells in AH were significantly higher than those in CSF. CONCLUSIONS: The majority of CD4+ lymphocytes in AH and CSF from patients with active VKH were activated memory cells, on which Fas antigen was also highly expressed. Although this Fas expression may not be an apoptosis-related phenomenon, accumulation of Fas+ memory T lymphocytes in AH and CSF probably reflects the immunopathologic mechanism of VKH.     

Deletion of the nef gene abrogates the ability of SIV smmPBj to induce acutely lethal disease in pigtail macaques

Simian immunodeficiency virus (SIV) infection in macaque species is typically associated with the development of a progressive immunodeficiency disease, similar to human AIDS, resulting in death of animals in months to years after infection. In contrast, a variant virus, termed SIVsmmPBj, induces an acute disease in macaques, resulting in death in 5 to 14 days after infection. Previously, we have shown that several viral determinants contribute to the pathogenesis of this disease. The present study was undertaken to evaluate the role of Nef in the pathogenesis of SIVsmmPBj-induced acute disease. A molecular clone of SIVsmmPBj was generated that contains a deletion in the nef coding region (PBj6.6 delta nef). Virus derived from this molecular clone was tested with the parental virus, PBj6.6, in replication studies in pigtail macaque and rhesus macaque peripheral blood mononuclear cells (PBMCs). In general, PBj6.6 delta nef displayed markedly reduced replication abilities when compared with PBj6.6; the only exception being in stimulated pigtail macaque PBMCs, where replication kinetics were nearly identical. In addition, PBj6.6 delta nef was unable to induce the proliferation of peripheral blood mononuclear cells (PBMCs) in vitro, a unique characteristic of acutely pathogenic SIVsmmPBj. Inoculation of this virus into pigtail macaques resulted in infection, but did not result in any detectable acute disease. These studies suggest that Nef is an important viral determinant in the pathogenesis of SIVsmmPBj-induced disease, and further suggest that Nef plays a significant role in viral replication in vivo.     

Fluorescein-labeled dextran concentration is increased in BAL fluid after ANTU-induced edema in rats

We identified the cell cycle status of CD34(+) cells of steady-state bone marrow (BM) and peripheral blood (PB) obtained from healthy volunteers, and those of apherasis PB samples collected from healthy donors who had been administered granulocyte colony-stimulating factor (G-CSF). More than 10% of CD34(+) cells in BM were in S+G2/M phase. In contrast, regardless of whether G-CSF treatment was performed, less than 2% of CD34(+) cells in PB were cycling. BM CD34(+) cells showed greater VLA-4 expression and adherence to stromal cells than PB CD34(+) cells. In addition, when cycling and dormant BM CD34(+) cells were analyzed separately, the cells in S+G2/M phase expressed more VLA-4 and adhered to the stromal cell monolayer more efficiently than the cells in G0/G1 phase. Furthermore, this adhesion of CD34(+) cells to the stromal cell layer was almost completely inhibited by anti-VLA-4 antibody. Taken together, these results suggest that CD34(+) progenitors in G0/G1 phase of the cell cycle differ from those in S+G2/M phase in adhesiveness mediated by VLA-4 in the hematopoietic microenvironment.     

c-myc and c-myb expression in acute myelogenous leukemia

Monoclonal antibodies and flow cytometry were used to detect the expression of c-myc and c-myb in the bone marrow (BM) and peripheral blood (PB) cells of patients with acute myelogenous leukemia (AML). The expression of neither gene was correlated with the percent blast cells in the BM or PB nor was there a correlation between c-myc and c-myb expression. A wide range of expression of each gene was found within each FAB type of AML. Patients who had a high proportion of leukemia cells expressing c-myb were less likely to respond to remission induction therapy than patients in whom a low proportion of cells expressed c-myb. This association appears to reflect an inverse relationship between the proportion of cells expressing c-myb and the sensitivity of leukemia cells to the killing effects of chemotherapy in vivo. Treatment outcome was unrelated to c-myc expression.     

Expression and functional significance of an activation-dependent epitope of the beta 1 integrins in chronic inflammatory diseases

The avidity of VLA integrins for their ligands can be increased by their transition to an active conformational state. This conformational change can be detected with a novel monoclonal antibody (mAb), termed 15/7, that recognizes an activation-dependent conformational epitope on the common beta 1 polypeptide of different VLA alpha beta 1 integrins. In an attempt to understand the possible role of the active conformational state of beta 1 integrins in vivo, we first investigated the expression of 15/7 epitope on T lymphocytes from patients with chronic inflammatory joint diseases. An enhanced expression of the 15/7 epitope was found in the synovial fluid (SF) T lymphocytes from these patients as compared to their peripheral blood (PB) T cells. The effect of different cytokines on the appearance of the 15/7 activation epitope in PB T lymphocytes was subsequently analyzed; interferon-gamma, interleukin-2 and, to a lower extent, tumor necrosis factor-alpha were able to induce an increased expression of the 15/7 epitope. This enhanced 15/7 expression correlated with a higher binding ability to fibronectin of cytokine-activated T cells. The presence of this activation epitope was detected in a small proportion of T lymphocytes scattered within inflammatory foci of synovial membrane from rheumatoid arthritis and thyroid glands from Hashimoto's chronic thyroiditis. We then analyzed the possible role of 15/7 epitope expression on cell adhesion in vitro. Immunofluorescence studies showed that the 15/7 epitope displayed a spot-like distribution, selectively decorating adhesive contacts of U-937 myelomonocytic cells attached to the 80 kDa proteolytic fragment of fibronectin (FN80). Furthermore, the anti-beta 1 15/7 mAb was able to induce both T lymphocyte, Jurkat and U-937 cellular binding and spreading on FN80. Altogether these results indicate that an activated conformation of beta 1 integrins is detected in vivo in lymphocyte infiltrates from chronic inflammatory conditions. The active conformations of beta 1 integrins are regulated by physiologic mediators such as cytokines, play an important role in cellular attachment and spreading, and appear to be involved in the development of inflammatory processes.     

Modulation of apoptosis with cytokines in B-cell chronic lymphocytic leukaemia

In B chronic lymphocytic leukaemia (B-CLL) non-proliferating peripheral blood (PB) B cells have a long life span in vivo. In cultures, these cells die spontaneously by apoptosis. Interleukin (IL) 4 inhibits spontaneous apoptosis (SA) and promotes survival of B-CLL B cells in vitro. No such effect is observed in PB B cells from normal healthy donors. The anti-apoptotic effect of IL4 is independent of mitogen-induced cell activation but depends on the concentration of IL4. The protective effect of IL4 is specific and it is significantly reduced or abolished with anti-IL4 antibody. Interferon (IFN)-gamma and alpha- IFN also protect B-CLL B cells from apoptosis in vitro. Sera from B-CLL patients have increased levels of IFN-gamma when compared with sera from healthy donors. In addition, B-cells in B-CLL express detectable levels of IFN-gamma mRNA. Other cytokines, namely ILl, IL2, IL6 and IL7 do not affect SA of B-CLL B cells. By contrast, IL5 and antibody to apolipoprotein-1 (APO- 1) receptor increase SA significantly and in a dose-dependent manner. Interleukin 4 protects B-CLL B cells from IL5-, anti(alpha) APO-1- and steroid-induced apoptosis. The mode of action of the cytokines inducing apoptosis or protecting B-CLL B cells from dying is largely unknown. Recently the bcl-2 proto-oncogene has been associated with prolonged cell survival. However, the involvement of bel-2 in spontaneous, cytokine-induced or steroid-induced apoptosis in B-CLL has been controversial. Some authors have reported down-regulation of bcl-2 protein expression in B-CLL B-cells undergoing SA or in steroid-treated cells with IL4 preventing this down-regulation. By contrast, others observed no significant loss of bcl-2 protein expression in steroid-, alpha-APO-1 - and IL5-treated cells when compared with untreated or fresh cells. Also, no correlation between bcl-2 protein expression and protection with IL4 has been reported. In conclusion, in B-CLL IL4, IFN-gamma and alpha-IFN promote the survival of the leukaemic cells. These cytokines may therefore be involved in the pathogenesis of the B-CLL.     

Cytokine-regulated expression of activated leukocyte cell adhesion molecule (CD166) on monocyte-lineage cells and in rheumatoid arthritis synovium

OBJECTIVE: To determine whether monocyte/macrophage expression of the CD6 ligand, activated leukocyte cell adhesion molecule (ALCAM) (CD166), is regulated by cytokines during inflammation in rheumatoid arthritis (RA). METHODS: We used flow cytometry to test whether cytokines present in rheumatoid synovium could regulate ALCAM cell surface expression on peripheral blood (PB) monocytes and RA synovial fluid (SF) macrophages, and we examined ALCAM expression in situ in RA synovium by immunofluorescence. RESULTS: The monocyte differentiation factors interleukin-3, macrophage colony-stimulating factor (M-CSF), and granulocyte-macrophage colony-stimulating factor augmented ALCAM expression on PB monocytes. ALCAM was expressed on monocyte-lineage cells in situ in inflamed synovium from patients with RA (9 of 9), but not in uninflamed synovium from patients with joint trauma (0 of 3). Furthermore, in vitro culture-induced ALCAM expression on PB monocytes and CD14+ RA SF cells was inhibited by an M-CSF neutralizing antibody. CONCLUSION: ALCAM expression on PB and SF monocytes/macrophages is enhanced by M-CSF.