Imatinib inhibits the in vitro development of the monocyte/macrophage lineage from normal human bone marrow progenitors.

The antileukaemic tyrosine kinase inhibitor, imatinib, has been reported to inhibit specifically the growth of bcr-abl expressing CML progenitors at levels of 0.1-5.0 microM, by blocking the ATP-binding site of the kinase domain of bcr-abl. Inhibition of the c-abl, platelet-derived growth factor receptor and stem cell factor receptor (c-kit) tyrosine kinases by imatinib has also been reported. Here, we demonstrate that imatinib significantly inhibits in vitro monocyte/macrophage development from normal bone marrow progenitors, while neutrophil and eosinophil development was less affected. Monocyte/macrophage inhibition was observed in semisolid agar and liquid cultures at concentrations of imatinib as low as 0.3 microM. The maturation of monocytes into macrophages was also found to be impaired following treatment of cultures with 1.0 microM imatinib. Imatinib blocked monocyte/macrophage development in cultures stimulated with and without M-CSF, suggesting that inhibition of the M-CSF receptor, c-fms, by imatinib was unlikely to be responsible. Imatinib may therefore have an inhibitory activity for other kinase(s) that play a role in monocyte/macrophage differentiation. This inhibition of normal monocyte/macrophage development was observed at concentrations of imatinib achievable pharmacologically, suggesting that imatinib or closely related derivatives may have potential for the treatment of diseases where monocytes/macrophages contribute to pathogenesis.

Increased expression of the CD45RO (memory) antigen on T cells in HIV-infected children.

Expression of the CD45RO putative memory cell antigen on CD4 (helper) and CD8 (cytotoxic/suppressor) lymphocytes of children born to HIV-infected women was investigated using the UCHL1 antibody. Significantly raised numbers of CD45RO+ CD8 lymphocytes were found in all nine of the infected children compared with uninfected and control children. Expression of CD45RO on CD4 lymphocytes was variable; absolute numbers were not increased, although the percentage was increased in four out of nine infected children. All the infected children except two (who had comparatively low numbers of CD45RO+ CD8 cells) were clinically well, which suggests that an increase in CD45RO+ CD8 cells may be indicative of a functionally active immune response against HIV.

The immunosuppressive compound 2-acetyl-4-tetrahydroxybutyl imidazole inhibits the allogeneic mixed lymphocyte reaction by sequestration of a recirculating subpopulation of T cells.

2-acetyl-4(5)-(1,2,3,4-tetrahydroxybutyl)imidazole (THI) is an immunosuppressive component of caramel food colouring that causes lymphopenia in mice and rats by an unknown mechanism. In this study we investigated some of the affects of THI on the murine immune system. Initially we showed that splenic T lymphocytes from mice treated with 50 mg/l THI in their drinking water were unable to launch a mixed lymphocyte reaction (MLR) against allogeneic stimulator cells, and had decreased and delayed interleukin-2 (IL-2) production. However, these T cells exhibited a normal proliferative response to concanavalin A (Con A), immobilized anti-CD3 monoclonal antibody (mAb) and anti-CD3 plus anti-CD28 mAb. Furthermore, the MLR response could be restored by the addition of IL-2 to the MLR culture. Homing studies using intravenous injection of fluorescence-labelled splenocytes showed that THI treatment decreased absolute numbers of labelled T and B lymphocytes in the blood and the spleen. Furthermore, these labelled cells reappeared in the blood and the spleen when mice were taken off THI, indicating that lymphocyte recirculation and splenic homing were modified reversibly by THI treatment. Cessation of THI treatment also resulted in a rapid reappearance of MLR responsiveness in the spleen, indicating that THI treatment does not functionally impair recirculating T cells. Collectively these data are compatible with the concept that a rapidly recirculating population of T cells, which produce IL-2 in an allogeneic MLR, are lost from the blood and spleen following THI treatment, and are sequestered in other, yet to be identified, tissues.

Prevention of autoimmunity by induction of cutaneous tolerance.

Autoimmune gastritis develops in 20-60% of BALB/c mice following thymectomy at 3 days after birth (3dnTx). Previously we identified the gastric H+/K+ ATPase as the causative autoantigen and mapped the immunoreactive T cell epitope to a carboxyl-terminal peptide on the gastric H+/K+ ATPase beta subunit. Here we show that autoimmune gastritis can be suppressed by immunizing 3dnTx mice through neonatal skin with the beta subunit peptide, in combination with the contact sensitizer TNCB. When spleen cells were transferred from suppressed mice to nude mice a proportion of recipient mice developed gastritis. These results indicate that pathogenic T cells were still present in the 3dnTx mice but the absence of gastritis indicates that their activity can be regulated following induction of cutaneous tolerance by immunizing through neonatal skin. We propose that cutaneous tolerance is induced through mediation of immature Langerhans cells in neonatal skin and that this tolerance prevented the autoreactivity of pathogenic T cells. This procedure will have implications for strategies to suppress autoimmunity.

Age-related ranges of memory, activation, and cytotoxic markers on CD4 and CD8 cells in children.

The expression of markers defining functional subpopulations on the surface of CD4 and CD8 cells changes with disease. To monitor these changes in children, it is important to establish the age-related normal changes in marker expression due to maturation of the immune system. We have studied the expression of several functionally important molecules on both CD4 and CD8 cells in 168 children (aged 0-122 months) using monoclonal antibodies and flow cytometry. Our results show that the percentage of CD4 cells decreases with age, while the CD8 percentage increases, resulting in a decrease in the CD4/CD8 ratio. The expression of CD45RO and CD29 increases with age, while CD45RA expression decreases, both on CD4 and CD8 cells. The expression of HLA-DR on both CD4 and CD8 cells, and of CD11a and CD57 on CD8 cells, is less clearly age dependent. The relationships between the marker percentages and age were not straightforward; the standard deviations and the skewness, as well as their mean values, varied as a function of age. The changes were modeled for each marker and age-specific centiles are presented.

CD4 and CD8 lymphocytes in diagnosis and disease progression of pediatric HIV infection.

Vertical infection with human immunodeficiency virus-1 (HIV-1) causes profound changes in the proportions of subpopulations of lymphocytes in the peripheral circulation. In this study the percentages in whole blood of CD4 and CD8 cells, and of immunologically important subpopulations, were measured in 19 HIV-infected children over periods of up to 4 years and compared to our recently published ranges for normal children of various ages. The rate of CD4 decline and of CD8 increase differed between clinically fast and slow progressors. On CD8 cells, cytotoxic, memory (CD11abright and CD45R0), and activation (HLA-DR) markers were raised soon after birth to levels outside the normal range, and compared favorably with HIV culture as a method for early diagnosis of HIV infection. Mean levels of naive (CD45RA) and memory (CD45R0, CD29) markers on CD4 cells became significantly altered after 48 months of age, suggesting that these are markers of more advanced disease. Despite different ages of enrollment into the study, in the cohort as a whole, the levels of the lymphocyte subpopulations studied changed consistently. Thus, their measurement could be useful both in the diagnosis and prognosis of HIV infection in individual children. This is the first report showing that lymphocyte subpopulation analysis can play a major role in the diagnosis of pediatric HIV infection.

Carcinogen-modified dendritic cells induce immunosuppression by incomplete T-cell activation resulting from impaired antigen uptake and reduced CD86 expression.

Exposure of the skin to environmental stimuli, such as chemical or physical carcinogens, modifies the local skin environment, including depletion of epidermal Langerhans' cells (LC). Any subsequent exposure of the LC-depleted skin to antigen results in the generation of antigen-specific tolerance. In this study we evaluated the antigen-bearing cells in the draining lymph nodes by capitalizing on the fluorescent nature of the contact sensitizer, fluorescein isothiocyanate (FITC). When FITC was applied to the skin of normal mice, two distinct populations of antigen-bearing cells were identified in the draining lymph nodes. They were classified as either FITChi or FITClo on the basis of their fluorescence intensity and thus the amount of antigen they internalized. Only FITClo cells were detected in the lymph nodes draining FITC-treated murine skin that had been depleted of epidermal LC by prior treatment with the complete carcinogen 9,10-dimethyl 1,2-benzanthracene (DMBA). Functional analysis of these cells revealed that the FITChi cells, but not the FITClo cells, induced antigen-specific T-cell proliferation. Further analysis of the FITClo cells from the DMBA-treated mice demonstrated that these cells had reduced levels of CD80 expression, had substantially reduced levels of CD86 expression and performed poorly as co-stimulator cells in an anti-CD3-mediated proliferative assay. Nonetheless these cells still induced early signs of T-cell activation and interleukin-12 production. Consequently the FITClo cells migrating from the LC-depleted skin, through a combination of reduced antigen presentation and reduced co-stimulatory activity, induced a state of unresponsiveness or anergy in the responder T cells in a similar manner to that observed when antigen presentation occurs in the absence of co-stimulation. We propose that these unresponsive, or anergic cells, account for the antigen-specific tolerance observed in these experiments.

Acquisition of immune function during the development of the Langerhans cell network in neonatal mice.

The immunological function of the Langerhans cell (LC) network in neonatal skin was examined by defining the development of cutaneous immunity relative to the structure, phenotype and function of the epidermal LC network in neonatal, juvenile and adult mice. Analysis of epidermal sheets showed the presence of major histocompatibility complex (MHC) II+, multilectin receptor DEC-205- cells within the epidermis of 3-day-old mice; both cell density and DEC-205 expression increased until day 14. When visualized with antibodies directed at MHC II, the network was poorly formed in 3- and 7-day-old mice, as there was a lower cell density and poor MHC II expression on dendritic processes, compared to mice at day14. Application of a fluorescent antigen to 3-day-old mice revealed that the LC were inefficient in transporting antigen to the draining lymph node. There was an improvement at day 7 and by day 14 comparable numbers of antigen carrying cells were detected in the lymph nodes of 6-week-old mice. The reduced antigen carriage in 3- and 7-day-old mice correlated with a poor contact sensitivity response. This was not simply due to failure to present antigen, but development of immunosuppression, as transfer of T cells from adult mice that were previously treated with antigen when they were 3 days old, to adult recipients resulted in antigen specific immunosuppression. Analysis of CD80 and CD86 expression showed that LC from day 3 skin expressed CD80, but not CD86 and application of antigen through this skin was inefficient in upregulating CD86. These findings indicate that when the neonatal LC network is poorly developed it is functionally immature and antigen applied through this 'functionally immature network' results in antigen specific immunosuppression.