Kelly L. Summers

Phenotypic Characterization of Five Dendritic Cell Subsets in Human Tonsils

Heterogeneous expression of several antigens on the three currently defined tonsil dendritic cell (DC) subsets encouraged us to re-examine tonsil DCs using a new method that minimized DC differentiation and activation during their preparation. Three-color flow cytometry and dual-color immunohistology was used in conjunction with an extensive panel of antibodies to relevant DC-related antigens to analyze lin(-) HLA-DR(+) tonsil DCs. Here we identify, quantify, and locate five tonsil DC subsets based on their relative expression of the HLA-DR, CD11c, CD13, and CD123 antigens. In situ localization identified four of these DC subsets as distinct interdigitating DC populations. These included three new interdigitating DC subsets defined as HLA-DR(hi) CD11c(+) DCs, HLA-DR(mod) CD11c(+) CD13(+) DCs, and HLA-DR(mod) CD11c(-) CD123(-) DCs, as well as the plasmacytoid DCs (HLA-DR(mod) CD11c(-) CD123(+)). These subsets differed in their expression of DC-associated differentiation/activation antigens and co-stimulator molecules including CD83, CMRF-44, CMRF-56, 2-7, CD86, and 4-1BB ligand. The fifth HLA-DR(mod) CD11c(+) DC subset was identified as germinal center DCs, but contrary to previous reports they are redefined as lacking the CD13 antigen. The definition and extensive phenotypic analysis of these five DC subsets in human tonsil extends our understanding of the complexity of DC biology.

Co-expression of the CD45RA and CD45RO antigens on T lymphocytes in chronic arthritis.

The site of T lymphocyte activation in chronic arthritis is unknown. Peripheral blood (PB) lymphocytes from chronic arthritis patients are in a ‘naïve’ or non‐activated state, as defined by expression of the CD45RA antigen and lack of HLA class II expression. In contrast, most synovial fluid (SF) T lymphocytes express a ‘memory’ or activated phenotype, as defined by the CD45RO antigen and high HLA class II expression. Following stimulation, naive cells lose CD45RA and gain CD45RO expression to become memory cells with a transitional stage of dual CD45RA, CD45RO antigen expression. To localize where this change in phenotype occurs we used dual colour immunofluorescence labelling to compare the percentage of dual CD45RA, CD45ROpositive T lymphocytes in PB and SF from chronic arthritic patients and from normal PB, assuming this population would be increased at the primary site of T lymphocyte activation. Expression of the intermediate and late activation marker. HLA‐DR, was also analysed using dual colour immunofluorescence labelling. The percentage of dual positive T lymphocytes was similar between arthritic PB, SF. and normal PB, as was the density of both CD45RA and CD45RO antigens. Thus, CD45 isoform expression did not indicate where T lymphocytes were activated. However, we identified a previously unreported population of CD45RA+ CD45RO+ HLA‐DR‐ T lymphocytes in arthritic and normal PB. In SF, this population was absent, but a substantial number of dual CD45RA, CD45RO‐positive HLA‐DR+ T lymphocytes were identified. This population would not be predicted by the current model of T lymphocyte activation. Division of T lymphocytes into functional groups on the basis of CD45 isoform expression is likely to be more complicated than previously thought. Based on our findings we propose an alternative model of T lymphocyte differentiation.

Dendritic cells in synovial fluid of chronic inflammatory arthritis lack CD80 surface expression

Dendritic cells (DC) act as potent primary antigen‐presenting cells in many immune responses and therefore may have a role in the initiation and perpetuation of the synovial inflammation in chronic inflammatory arthritis. To examine their function, it is important to isolate fresh DC from arthritic joints without aberrant activation. We have developed a technique using minimal cell manipulation to isolate DC from the synovial fluid of chronic arthritic patients. Using this method, DC were shown to be potent allostimulatory cells, with 63–90% of cells lacking lineage‐specific markers (lin‐), but positive for MHC class II molecules. Two morphologically distinct populations of these cells were identified in 10 out of 13 DC preparations. Both populations expressed CD40, intercellular adhesion molecule‐1 (ICAM‐1), ICAM‐2, ICAM‐3 and leucocyte function associated antigen‐3 (LFA‐3), but the predominant population, which was larger and more typical of cultured blood DC, had a higher density of these antigens compared with the minor population, which were smaller and morphologically similar to lymphocytes. Two new MoAbs which label activated human blood DC, HB15 (CD83) and CMRF‐44, were tested. CD83 labelled very weakly or not at all, whereas CMRF‐44 was positive on the larger cells only. Likewise, the costimulator molecule, B7/BB1 (CD80), was not detected on the surface of either synovial lin‐ cell population, reverse transcriptase polymerase chain reaction (RT‐PCR) showed little or no CD80 mRNA, and no binding of the CTLA‐4Ig fusion protein was found. These results suggest that synovial DC are not, despite the inflammatory environment, in a fully activated state.

Noncooperative Metalation of Metallothionein 1a and Its Isolated Domains with Zinc

Mammalian metallothioneins (MTs) are a family of small cysteine-rich proteins capable of binding 7 Zn(2+) or Cd(2+) ions into two distinct domains: an N-terminal β-domain that binds 3 Zn(2+) or Cd(2+) and a C-terminal α-domain that binds 4 Zn(2+) or Cd(2+). MT has been implicated in a number of physiological functions, including metal ion homeostasis, toxic metal detoxification, and as a protective agent against oxidative stress. Conventionally, MT has been understood to coordinate metal ions in a cooperative fashion. Under this mechanism of metalation, the only species of biological relevance would be the metal-free (apo-) form of the protein and the fully metalated (holo-) form of the protein. However, an increasing body of evidence suggests that metalation occurs in a noncooperative manner. If this latter mechanism is correct, then partially metalated forms of the protein will be stable and able to take part in cellular chemistry. We report in this paper conclusive evidence that shows that biologically essential zinc binds to MT in a noncooperative manner. In addition, we report for the first time the stability of a Zn(5)-MT species. The implications of these findings are discussed in terms of the mechanism of metalation.

Synovial fluid transforming growth factor β inhibits dendritic cell–T lymphocyte interactions in patients with chronic arthritis

OBJECTIVE To examine whether rheumatoid synovial fluid (SF) inhibits dendritic cell (DC) expression of the CD80 and CD86 costimulator molecules and contributes to SF T lymphocyte hyporesponsiveness. METHODS Cell-free rheumatoid SF was tested for its effect on DC-stimulated autologous/allogeneic mixed lymphocyte reactions and for its effect on DC surface antigen expression, as assessed by flow cytometry. Blocking monoclonal antibodies were used to identify the SF cytokines that inhibited DC-T lymphocyte interactions. RESULTS Low concentrations of SF (2.5%) could inhibit DC-mediated autologous and allogeneic T lymphocyte proliferation. This inhibitory effect could be reversed by neutralizing transforming growth factor beta (TGFbeta) and interleukin-2 (IL-2), but not by IL-12, in the SF. Hyaluronic acid, IL-6, IL-10, and tumor necrosis factor alpha were not associated with SF inhibition. In vitro culture alone and crosslinking with the CD40 ligand up-regulated DC CD80/CD86 expression and costimulator function, and this was not affected by inclusion of SF. In the presence of SF, DC clustered with autologous T lymphocytes showed decreased CD80 and CD86 expression, and variable CD80/CD86 decreases were observed on DC clustered with allogeneic T lymphocytes. CONCLUSIONS TGFbeta in SF appears to suppress T lymphocyte function, which may affect both signaling to DC and the induction of DC costimulator function.

Maternal, umbilical arterial and umbilical venous 25‐hydroxyvitamin D and adipocytokine concentrations in pregnancies with and without gestational diabetes

Gestational diabetes mellitus (GDM) has been associated with inflammation as well as Vitamin D insufficiency. While Vitamin D has anti‐inflammatory properties, relationships between Vitamin D and inflammatory markers remain unexplored in GDM. Therefore, this case‐–control study investigated adipocytokine and Vitamin D [25(OH)D] concentrations and correlations in GDM and control women, as well as their neonates.

Cysteine accessibility during As3+ metalation of the α- and β-domains of recombinant human MT1a.

Metallothionein is a ubiquitous metal binding protein that plays an important role in metal ion homeostasis and redox chemistry within cells. Mammalian metallothioneins bind a wide variety of metals including the metalloid As3+ in two domains (β and α) connected by a short linker sequence. Three As3+ bind in each domain for a total of 6 As3+ per protein. In recombinant human metallothionein (rh-MT1a) each As3+ binds three cysteine residues to form As3Cys9(CysSH)2-α-rhMT1a in the 11 Cys α-domain and As3Cys9-β-rhMT1a in the 9 Cys β-domain. This means that there should be 2 free cysteines in the α-domain but no free cysteines in the β-domain. By using benzoquinone, the number and relative accessibility of the free cysteinyl thiols during the metalation reactions were determined. The electrospray ionization mass spectrometry (ESI-MS) data confirmed that each As3+ binds using exactly 3 cysteine thiols and showed that there was a significant difference in the reactivity of the free cysteines during the metalation reaction. After a reaction with two molar equivalents of As3+ to form As2Cys6(CysSH)3-αβ-rhMT1a, the remaining 3 Cys in the 9 Cys β-domain were far less reactive than those in the α-domain. Molecular dynamics calculations for the metalation reactions with As3+ measured by ESI-MS allowed an interpretation of the mass spectral data in terms of the relative location of the cysteine thiols that were not involved in As3+ coordination. Together, these data provide insight into the selection of a specific cysteinyl thiol by the incoming metals during the stepwise metalation of metallothioneins.

Diabetic Ketoacidosis Elicits Systemic Inflammation Associated with Cerebrovascular Endothelial Cell Dysfunction

To determine if the DKA‐induced inflammation in juvenile mice provokes activation and dysfunction of CVECs.

Probiotic Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 May Help Downregulate TNF-Alpha, IL-6, IL-8, IL-10 and IL-12 (p70) in the Neurogenic Bladder of Spinal Cord Injured Patient with Urinary Tract Infections: A Two-Case Study

The management of urinary tract infection (UTI) in individuals with spinal cord injury (SCI) continues to be of concern, due to complications that can occur. An emerging concept that is a common underlying pathophysiological process is involved, wherein pathogens causing UTI have a role in inflammatory progression. We hypothesized that members of the commensal flora, such as lactobacilli, may counter this reaction through anti-inflammatory mediation. This was assessed in a pilot two-patient study in which probiotic Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri were administered to one patient and placebo to another, both along with antibiotics to treat acute UTI. Urinary TNF-alpha was significantly downregulated (P = .015) in the patient who received the probiotic and who used intermittent catheterization compared with patient on placebo and using an indwelling catheter. The extent to which this alteration resulted in improved well-being in spinal cord injured patients remains to be determined in a larger study.

Differential Contributions of APC Subsets to T Cell Activation in Nonobese Diabetic Mice

Despite the pivotal role of dendritic cells (DC) in shaping immunity, little is known about their functionality in type 1 diabetes. Moreover, due to the paucity of DC in vivo, functional studies have relied largely upon in vitro-expanded cells to elucidate type 1 diabetes-associated functional abnormalities. In this study, we provide a comprehensive analysis of the functional capabilities of in vivo-derived DC subsets from NOD mice by comparing DC to other NOD APC types and to DC from autoimmune-resistant strains. NOD DC closely resemble those from nonautoimmune strains with respect to costimulation and cytokine production. The exception is the CD8α+CD11b−DC subset which is numerically reduced in NOD spleens, but not in the pancreatic lymph nodes, while DC from both tissues produce little IL-12 in this strain. This defect results in unusual deferral toward macrophage-derived IL-12 in NOD mice; NOD macrophages produce aberrantly high IL-12 levels that can overcompensate for the DC defect in Th1 polarization. APC subset use for autoantigen presentation also differs in NOD mice. NOD B cells overshadow DC at activating islet-reactive T cells, whereas DC and B cells in NOD-resistant mice are functionally comparable. Differential involvement of APC subsets in T cell activation and tolerance induction may prove to be a crucial factor in the selection and expansion of autoreactive T cells.