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1.  Macrophages exposed continuously to lipopolysaccharide and other agonists that act via toll-like receptors exhibit a sustained and additive activation state 
BMC Immunology  2001;2:11.
Macrophages sense microorganisms through activation of members of the Toll-like receptor family, which initiate signals linked to transcription of many inflammation associated genes. In this paper we examine whether the signal from Toll-like receptors [TLRs] is sustained for as long as the ligand is present, and whether responses to different TLR agonists are additive.
RAW264 macrophage cells were doubly-transfected with reporter genes in which the IL-12p40, ELAM or IL-6 promoter controls firefly luciferase, and the human IL-1β promoter drives renilla luciferase. The resultant stable lines provide robust assays of macrophage activation by TLR stimuli including LPS [TLR4], lipopeptide [TLR2], and bacterial DNA [TLR9], with each promoter demonstrating its own intrinsic characteristics. With each of the promoters, luciferase activity was induced over an 8 hr period, and thereafter reached a new steady state. Elevated expression required the continued presence of agonist. Sustained responses to different classes of agonist were perfectly additive. This pattern was confirmed by measuring inducible cytokine production in the same cells. While homodimerization of TLR4 mediates responses to LPS, TLR2 appears to require heterodimerization with another receptor such as TLR6. Transient expression of constitutively active forms of TLR4 or TLR2 plus TLR6 stimulated IL-12 promoter activity. The effect of LPS, a TLR4 agonist, was additive with that of TLR2/6 but not TLR4, whilst that of lipopeptide, a TLR2 agonist, was additive with TLR4 but not TLR2/6. Actions of bacterial DNA were additive with either TLR4 or TLR2/6.
These findings indicate that maximal activation by any one TLR pathway does not preclude further activation by another, suggesting that common downstream regulatory components are not limiting. Upon exposure to a TLR agonist, macrophages enter a state of sustained activation in which they continuously sense the presence of a microbial challenge.
PMCID: PMC58839  PMID: 11686851
2.  HIV infection and aging: enhanced Interferon- and Tumor Necrosis Factor-alpha production by the CD8+ CD28- T subset 
BMC Immunology  2001;2:10.
T cells from HIV+ and aged individuals show parallels in terms of suppressed proliferative activity and interleukin-2 (I1-2) production and an increased number of CD8+ CD28- T cells. In order to compare cytokine production from T cells from these two states, CD4+ and CD8+ T cells from HIV+ aged, and normal young donors (controls) were monitored for cytokine production by flow cytometry, quantitative PCR and ELISA upon activation by PMA and anti-CD3. In addition, the CD8+ T cell subsets CD28+ and CD28- from the HIV+ and the aged groups were evaluated for cytokine production by flow cytometry, and compared with those from young controls.
Flow cytometric analysis indicated that CD8+ T cells from both HIV+ and aged donors showed an increase of approximately 2–3 fold over controls in percentage of cells producing inflammatory cytokines IFN-γ and TNF-α. Similar analysis also revealed that the production of interleukins-4,6 and 10, production was very low (1–2% of cells) and unchanged in these cells. Quantitative PCR also showed a substantial increase (4–5 fold) in IFN-γ and TNF-α mRNA from HIV+ and aged CD8+ T cells, as did ELISA for secreted IFN-γ and TNF-α (2.3–4 fold). Flow cytometric analysis showed that the CD8+ CD28- T cell subset accounts for approximately 80–86% of the IFN-γ and TNF-α production from the CD8+ subset in the aged and HIV+ states. The CD4+ T cell, while not significantly changed in the HIV+ or aged states in terms of IFN-γ production, showed a small but significant increase in TNF-α production in both states.
Our data appear compatible with physiologic conditions existing in HIV+ and aged individuals, i.e. elevated serum levels and elevated CD8+ T cell production of IFN-γ and TNF-α. Thus, the capacity for increased production of cytokines IFN-γ and TNF-α in the aged individual by the dominant CD8+ CD28- subset may have a profound influence on the clinical state by aggravating inflammatory pathologies such as rheumatoid arthritis, and possibly Alzheimer's disease and Crohn's disease. In AIDS, these cytokines may contribute to wasting and cachexia. We theorize that the predominant phenotypic change to the cytotoxic CD8+ CD28- T cell subsets in both the HIV+ and the aged states may reflect a natural "endpoint" in CD8+ T cell differentiation induced after a lifetime of immune activity (toward viruses, etc) in the aged, and after a massive accelerated response to HIV in the HIV-positive individual.
PMCID: PMC59583  PMID: 11696237
3.  Improvements to parallel plate flow chambers to reduce reagent and cellular requirements 
BMC Immunology  2001;2:9.
The parallel plate flow chamber has become a mainstay for examination of leukocytes under physiologic flow conditions. Several design modifications have occurred over the years, yet a comparison of these different designs has not been performed. In addition, the reagent requirements of many designs prohibit the study of rare leukocyte populations and require large amounts of reagents.
In this study, we evaluate modifications to a newer parallel plate flow chamber design in comparison to the original parallel plate flow chamber described by Lawrence et al. We show that modifications in the chamber size, internal tubing diameters, injection valves, and a recirculation design may dramatically reduce the cellular and reagent requirements without altering measurements.
These modifications are simple and easily implemented so that study of rare leukocyte subsets using scarce or expensive reagents can occur.
PMCID: PMC56996  PMID: 11580861
4.  Role of CD28/B7 costimulation and IL-12/IL-10 interaction in the radiation-induced immune changes 
BMC Immunology  2001;2:8.
The present paper aims at studying the role of B7/CD28 interaction and related cytokine production in the immunological changes after exposure to different doses of ionizing radiation.
The stimulatory effect of low dose radiation (LDR) on the proliferative response of lymphocytes to Con A was found to require the presence of APCs. The addition of APCs obtained from both low- and high-dose-irradiated mice to splenic lymphocytes separated from low-dose-irradiated mice caused stimulation of lymphocyte proliferation. B7-1/2 expression on APCs was up-regulated after both low and high doses of radiation. There was up-regulation of CD28 expression on splenic and thymic lymphocytes after LDR and its suppression after high dose radiation (HDR), and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) expression showed changes in the opposite direction. IL-12 secretion by macrophages was stimulated after both low and high doses of radiation, but IL-10 synthesis by splenocytes was suppressed by low dose radiation and up-regulated by high dose radiation.
The status of CD28/CTLA-4 expression on T lymphocytes in the presence of up-regulated B7 expression on APCs determined the outcome of the immune changes in response to radiation, i.e., up-regulation of CD28 after LDR resulted in immunoenhancement, and up-regulation of CTLA-4 associated with down-regulation of CD28 after HDR led to immunosuppression. Both low and high doses of radiation up-regulated B7-1/2 expression on APCs. After LDR, the stimulated proliferative effect of increased IL-12 secretion by APCs, reinforced by the suppressed secretion of IL-10, further strengthened the intracellular signaling induced by B7-CD28 interaction.
PMCID: PMC48143  PMID: 11532194
5.  Alterations induced by chronic stress in lymphocyte subsets of blood and primary and secondary immune organs of mice 
BMC Immunology  2001;2:7.
The immune system is particularly sensitive to stress. Although acute stress generally has positive effects, chronic stress typically provokes immunosuppression. The elucidation of the mechanisms involved in immunosuppression are of interest for the design of therapeutic approaches to avoid the appearance of stress disorders. This study aimed to investigate chronic stress-induced alterations on lymphocyte subset distribution and percentages. The experiments were performed with C57BL/6 mice subjected to chronic immobilization stress.
Stress caused a marked increase in apoptosis inside the thymus, and a reduction in the total number of thymocytes. Furthermore, the proportion of immature thymocytes declined significantly suggesting that the increased apoptosis mainly affected cells of immature phenotype. In blood, the total number of lymphocytes diminished but not all lymphocyte populations showed the same tendency: while the relative proportion of B cells declined slightly, the relative proportion of circulating CD3+ cells, and particularly some T cell subsets showing an immature phenotype (CD3+PNA+), increased under stress. The spleen and lymph nodes show a marked reduction in cellularity, but the relative proportion of T cells increased, while no change or only a slight reduction was observed in the relative proportion of B cells. Similarly, the relative proportion of T cells increased in bone marrow.
Detailed data on the alterations of lymphoid cell subsets occurring under immobilization stress, both in the bloodstream and in different lymphoid tissues, are obtained. In general, T cells are more affected than B cells and, in particular, a marked increase in the percentage of a subset of circulating PNA+CD3+ T cells is observed.
PMCID: PMC37547  PMID: 11518541
6.  Flow cytometric assessment of the reactivity of a panel of monoclonal antibodies (mAb) against two populations of human dendritic cells (DC) 
BMC Immunology  2001;2:6.
The identification of antigens on human DC has been a very difficult and elusive task because of the lack of appropriate reagents. Therefore, we evaluated by flow cytometry a panel of mAb that recognize antigens on human DC, aiming to determine the kinetics of DC antigen expression at 7, 14, 21 and 28 days in (i) Dermal DC like cells (Mo-DC) and (ii) Langerhans cell like DC (Mo-LC). In addition we aimed to identify markers for DC subpopulations.
It was found at day 7, that mAb BG6, HP-F1, BU10, RFD-1, CMRF-44 recognized <20% of Mo-DC. In contrast, 7H5, ZM3.8, CDlb/c, 55K-2, MMR1.16, MMR190.BB3 and L25 reacted with >50% of Mo-DC. Moreover, 7H5, ZM3.8, CMRF-56, CDlb/c, 55K-2, MMR1.16, MMR190.BB3 and L25 showed increased MFI reactivity against Mo-DC. mAb BG6, BU10 and CMRF-44 recognized <20% Mo-LC while RFD-1 reacted with 21% of Mo-LC. In contrast, HP-F1 showed 87% of Mo-LC positive. Also, 7H5, ZM3.8, RFD-7, MR15-2, CDlb/c, 55K-2, MMR1.16, MMR190.BB3 and L25 reacted with >50% of Mo-LC. The increase in % of positive cells was paralleled by MFI increases.
At day 14, fourteen mAb recognized >50% of the Mo-DC, while five recognized 20-50% of Mo-DC. BG6 reacted with 7% of the Mo-DC. Nineteen mAb recognized >48% of Mo-LC while BG6 had negative reactivity.
At day 21 and 28, all mAb reacted with >20% of Mo-DC and yielded a significant MFI with Mo-DC. Also nineteen mAb yielded significant MFI with Mo-LC while RFD-7 did not.
The immunophenotyping assays demonstrated differences between the two DC populations as well as variations in the reactivity of the mAb at diverse time points, suggesting the existence of subpopulations within the Mo-DC and Mo-LC.
PMCID: PMC37353  PMID: 11504561
7.  Peptide binding characteristics of the non-classical class Ib MHC molecule HLA-E assessed by a recombinant random peptide approach 
BMC Immunology  2001;2:5.
Increasing evidence suggests that the effect of HLA-E on Natural Killer (NK) cell activity can be affected by the nature of the peptides bound to this non-classical, MHC class Ib molecule. However, its reduced cell surface expression, and until recently, the lack of specific monoclonal antibodies hinder studying the peptide-binding specificity HLA-E.
An in vitro refolding system was used to assess binding of recombinant HLA-E to either specific peptides or a nonamer random peptide library. Peptides eluted from HLA-E molecules refolded around the nonamer library were then used to determine a binding motif for HLA-E. Hydrophobic and non-charged amino acids were found to predominate along the peptide motif, with a leucine anchor at P9, but surprisingly there was no methionine preference at P2, as suggested by previous studies.
Compared to the results obtained with rat classical class Ia MHC molecules, RT1-A1c and RT1-Au, HLA-E appears to refold around a random peptide library to reduced but detectable levels, suggesting that this molecule's specificity is tight but probably not as exquisite as has been previously suggested. This, and a previous report that it can associate with synthetic peptides carrying a viral sequence, suggests that HLA-E, similar to its mouse counterpart (Qa-1b), could possibly bind peptides different from MHC class I leader peptides and present them to T lymphocytes.
PMCID: PMC33820  PMID: 11432755
8.  A conditional form of Bruton's tyrosine kinase is sufficient to activate multiple downstream signaling pathways via PLC Gamma 2 in B cells 
BMC Immunology  2001;2:4.
Bruton's tyrosine kinase (Btk) is essential for B cell development and function. Mutations of Btk elicit X-linked agammaglobulinemia in humans and X-linked immunodeficiency in the mouse. Btk has been proposed to participate in B cell antigen receptor-induced signaling events leading to activation of phospholipase C-γ2 (PLCγ2) and calcium mobilization. However it is unclear whether Btk activation is alone sufficient for these signaling events, and whether Btk can activate additional pathways that do not involve PLCγ2. To address such issues we have generated Btk:ER, a conditionally active form of the kinase, and expressed it in the PLCγ2-deficient DT40 B cell line.
Activation of Btk:ER was sufficient to induce multiple B cell signaling pathways in PLCγ2-sufficient DT40 cells. These included tyrosine phosphorylation of PLCγ2, mobilization of intracellular calcium, activation of extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways, and apoptosis. In DT40 B cells deficient for PLCγ2, Btk:ER activation failed to induce the signaling events described above with the consequence that the cells failed to undergo apoptosis.
These data suggest that Btk:ER regulates downstream signaling pathways primarily via PLCγ2 in B cells. While it is not known whether activated Btk:ER precisely mimics activated Btk, this conditional system will likely facilitate the dissection of the role of Btk and its family members in a variety of biological processes in many different cell types.
PMCID: PMC32313  PMID: 11410123
9.  Requirements for activation and RAFT localization of the T-lymphocyte kinase Rlk/Txk 
BMC Immunology  2001;2:3.
The Tec family kinases are implicated in signaling from lymphocyte antigen receptors and are activated following phosphorylation by Src kinases. For most Tec kinases, this activation requires an interaction between their pleckstrin homology (PH) domains and the products of phosphoinositide 3-Kinase, which localizes Tec kinases to membrane RAFTs. Rlk/Txk is a Tec related kinase expressed in T cells that lacks a pleckstrin homology domain, having instead a palmitoylated cysteine-string motif. To evaluate Rlk's function in T cell receptor signaling cascades, we examined the requirements for Rlk localization and activation by Src family kinases.
We demonstrate that Rlk is also associated with RAFTs, despite its lack of a pleckstrin homology domain. Rlk RAFT association requires the cysteine-string motif and is independent of PI3 Kinase activity. We further demonstrate that Rlk can be phosphorylated and activated by Src kinases, leading to a decrease in its half-life. A specific tyrosine in the activation loop of Rlk, Y420, is required for phosphorylation and activation, as well as for decreased stability, but is not required for lipid RAFT association. Mutation of this tyrosine also prevents increased tyrosine phosphorylation of Rlk after stimulation of the T cell receptor, suggesting that Rlk is phosphorylated by Src family kinases in response to T cell receptor engagement.
Like the other related Tec kinases, Rlk is associated with lipid RAFTs and can be phosphorylated and activated by Src family kinases, supporting a role for Rlk in signaling downstream of Src kinases in T cell activation.
PMCID: PMC31577  PMID: 11353545
10.  Changes in human lymphocyte subpopulations in tonsils and regional lymph nodes of human head and neck squamous carcinoma compared to control lymph nodes 
BMC Immunology  2001;2:2.
Lymphoid tissues constitute basic structures where specific immune responses take place. This leads to the development of germinal centres (GCs), migration of cells and the generation of memory cells. Here, we have compared human tumour reactive lymph nodes and tonsils with control lymph nodes.
The study by flow cytometry shows that in control lymph nodes the majority of cells were naive T-lymphocytes (CD45RA+/CD7+). In reactive nodes, although the percentage of CD45RO+ T cells remains constant, there is an increase in the number of B-lymphocytes, and a reduction in naive T cells. The percentage of cells expressing CD69 was similar in reactive nodes and in controls. In both cases, we have found two populations of B cells of either CD69- or CD69dull. Two populations of T cells, which are either negative for CD69 or express it in bright levels (CD69bright), were also found.
The analysis of tissue sections by confocal microscopy revealed differences between control, tonsils and tumor reactive lymph nodes. In control lymph nodes, CD19 B cells are surrounded by a unique layer of CD69bright/CD45RO+ T cells. GCs from tonsils and from tumour reactive nodes are mainly constituted by CD19 B cells and have four distinct layers. The central zone is composed of CD69- B cells surrounded by CD69bright/CD45RO+ T cells. The mantle region has basically CD69dull B-lymphocytes and, finally, there is an outer zone with CD69-/CD45RO+ T cells.
Human secondary lymphoid organs react with an increase in the proportion of B lymphocytes and a decrease in the number of CD45RA+ T cells (naive). In tonsils, this is due to chronic pathogen stimulation, whereas in lymph nodes draining head and neck carcinomas the reaction is prompted by surrounded tumors. During this process, secondary lymphoid organs develop secondary follicles with a special organization of T and B cells in consecutive layers, that are described here by confocal microscopy. This pattern of cellular distribution may suggest a model of cell migration into the secondary lymphoid follicles.
PMCID: PMC31349  PMID: 11316463
11.  In vitro production of peroxynitrite by haemocytes from marine bivalves: C-ELISA determination of 3-nitrotyrosine level in plasma proteins from Mytilus galloprovincialis and Crassostrea gigas 
BMC Immunology  2001;2:1.
Peroxynitrite is increasingly proposed as a contributor to defence system in marine bivalve. It can be formed by combinaison of superoxide and nitric oxide, and can react with tyrosine residues of proteins giving rise to 3-nitrotyrosine.
The present article describes a competitive ELISA for the measurement of 3-nitrotyrosine contents of plasma proteins from marine bivalves by means of a monoclonal anti 3-nitrotyrosine antibody mouse IgG.
This assay is sensitive enough to determine the amounts of 3-nitrotyrosine in plasma proteins from one animal only.
Using the C-ELISA, we have shown that the phagocytosis of zymosan particles increased the 3-nitrotyrosine levels of plasma proteins from mussel M. galloprovincialis and oyster C. gigas 5.8 and 7.5 times respectively.
PMCID: PMC31348  PMID: 11231884

Results 1-11 (11)