Elling Ulvestad

Population-Based Hematologic and Immunologic Reference Values for a Healthy Ugandan Population

ABSTRACT To assess the validity of the reference values for hematologic and immunologic indices currently used in Africa, we evaluated blood samples from 3,311 human immunodeficiency virus (HIV)-negative Ugandans aged 1 week to 92 years. Erythrocyte, hemoglobin, and hematocrit levels and mean corpuscular volume all significantly increased with age (P < 0.001) and were independent of gender until the age of 13 years, after which the levels were higher in males than in females (P < 0.001). White blood cell, neutrophil, lymphocyte, basophil, and monocyte counts significantly declined with age until the age of 13 years (P < 0.001), with no differences by gender, while platelet counts declined with age (P < 0.001) and showed differences by gender only among adults older than age 24 years. CD4+- and CD8+-cell counts declined with age until the age of 18 years; thereafter, females had higher counts than males. The absolute values for many of these parameters differed from those reported for populations outside Africa, suggesting that it may be necessary to develop tables of reference values for hematologic and immunologic indices specific for the African population. This may be particularly important with regard to CD4+-cell counts among children because significant differences in absolute and percent CD4+-cell counts exist between the values for Western populations and the values for the population evaluated in our study. These differences could influence the decision to initiate antiretroviral therapy among children infected with HIV.

Human microglial cells have phenotypic and functional characteristics in common with both macrophages and dendritic antigen-presenting cells.

Resting microglia comprise up to 13% of the cells in human central nervous system (CNS) white matter. Their large number and dendritic morphology make them ideally suited to survey the CNS for noxious stimuli. Upon activation microglia gradually lose dendritic processes and transform into typical phagocytic macrophages. Microglia have been implicated as the main antigen presenting cell within the CNS, and appear to be of central importance as effectors and regulators of demyelination. To further characterize the capacity for immune reactivity within the human CNS, we have studied several characteristics of microglia, both in situ and in vitro. We find that human microglia have ultrastructural, phenotypic (CD11c, CD68, acid phosphatase), and functional (FcR and CR mediated phagocytosis) properties typical for cells of the monocyte lineage. Our data indicate that microglia also have properties in common with dendritic antigen‐presenting cells. Electron microscopy studies show extended dendritic cell processes on cultured microglia, and microglia are, like dendritic cells, negative for the monocyte markers nonspecific esterase, endogenous peroxidase, CD14, and RFD7. Microglia constitutively express HLA‐DR in situ, and express the dendritic cell marker RFD1 upon activation. Coculturing of microglia with CD4+ T cells results in clustering of T cells around microglia and initiation of a mixed lymphocyte reaction, both distinguishing features of dendritic cells. These functional properties of microglia may be of importance for the maintenance of an immunologic response in the CNS, an organ where dendritic cells, in contrast to other organs, have not previously been identified. J. Leukoc. Biol. 56: 732–740; 1994.

IL‐6: an early marker for outcome in acute ischemic stroke

Objectives –  Inflammation plays an important role in the pathophysiology of stroke. We correlated interleukin (IL)‐6, IL‐10, C‐reactive protein (CRP) and T‐lymphocyte subtype levels in acute ischemic stroke patients with stroke volume and clinical outcome.

Reactive microglia in multiple sclerosis lesions have an increased expression of receptors for the Fc part of IgG

Receptors for the Fc part of IgG, FcRI (CD64), FcRII (CD32), and FcRIII (CD16) were studied by indirect immunoperoxidase staining of cryostat sections from normal and multiple sclerosis (MS) brains. Microglia in the parenchyma of normal white matter had a dendritic morphology, and were weakly stained by monoclonal antibodies (mAbs) to FcRI, FcRII, and FcRIII. In active MS lesions reactive microglia were strongly stained by the mAbs 32.2 (FcRI), IV-3 (FcRII), and 3G8 (FcRIII). Perivascular macrophages were stained by all anti-FcR mAbs in both normal white matter and in MS lesions, whereas endothelial cells were stained by the anti-FcRIII mAb only. The FcR on microglia and perivascular macrophages may be of functional importance in antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, and local immunoregulation. FcR on endothelium may be of importance in binding and transportation of immune complexes into the CNS. FcR mediated functions may consequently be highly relevant to the pathogenesis of MS.

Fc receptors for IgG on cultured human microglia mediate cytotoxicity and phagocytosis of antibody-coated targets.

We have utilized surgically resected human central nervous system (CNS) tissue to determine the expression and functions of Fc receptors (FcyR) on individual cell types found within the CNS. We observed all three classes of FcyR on microglial cells in situ and in vitro, but not on astrocytes or oligodendrocytes. Incubation of cultured microglia with immune complexes (antibody-coated red blood cells) induced phagocytosis, antibody-dependent cell-mediated cytotoxicity (ADCC), and oxidative bursts. We also found that microglia have the capability to produce T cell stimulatory soluble mediators after FcyR crosslinking. These functional responses were enhanced by pretreatment of the microglia with interferon-y (IFN-y). Our results implicate microglial effector responses triggered by interaction of FcyR with opsonized antigens as potential mediators of tissue injury within the CNS. Such injury may be particularly applicable to multiple sclerosis, an inflammatory demyelinating disease characterized by intrathecal production of immunoglobulins and cytokines.

Phenotypic Differences between Human Monocytes/Macrophages and Microglial Cells Studied In Situ and In Vitro

This report describes a phenotypic differentiation pattern conceived to distinguish invading monocytes from resident microglia in frozen and formalin-fixed human CNS. Phagocytic cells in normal and diseased CNS (multiple sclerosis and encephalitis) were studied immunohistochemically with a panel of antibodies, and phenotypic characteristics were compared with cultured monocytes/macrophages and microglia. Monocytes/macrophages were positive for the markers nonspecific esterase, myeloperoxidase, L1, lysozyme, RFD7, and CD 14, whereas microglia were negative for the same markers. Both populations of cells were positive for CD11c and CD68. Our results indicate that invading monocytes/macrophages mainly have a perivascular location in active multiple sclerosis lesions, whereas invading monocytes/macrophages also infiltrate the parenchyma in acute inflammatory CNS diseases such as in encephalitis.

Favourable response to therapy with the anti-CD20 monoclonal antibody rituximab in primary chronic cold agglutinin disease

The ‘primary’ form of chronic cold agglutinin disease is a clonal B‐cell lymphoproliferative disorder that is notoriously difficult to treat with drugs, including corticosteroids, alkylating agents, alpha‐interferon and purine analogues. We performed a small, open, uncontrolled, prospective study to evaluate the effect of therapy with the monoclonal anti‐CD20 antibody rituximab. Six patients with clonal CD20+κ+ B‐cell proliferation received seven courses of rituximab 375 mg/m2, d 1, 8, 15, and 22. One patient achieved a complete response. Four partial responses were observed, including a response to re‐treatment in one patient. Two patients were categorized as non‐responders. Haemoglobin levels increased by a median of 4·1 g/dl in the total group and 4·7 g/dl in the responders, who also experienced a substantial improvement of clinical symptoms. The treatment was well tolerated. We discuss the effect of rituximab therapy compared with other treatment options, and try to explain why two individual patients did not respond. Despite the small numbers, the results are very encouraging. Further studies of rituximab therapy for chronic cold agglutinin disease are warranted.

IL-10 PRODUCTION BY ADULT HUMAN DERIVED MICROGLIAL CELLS

Microglia, a population of central nervous system (CNS) macrophages, have been demonstrated to support immune accessory and effector functions in the CNS. Numerous studies support the role of microglia in CNS development and pathology, where activation of microglia is consistently noted. The current study investigated microglial immune functions under basal and activation conditions and assessed the ability of interleukin-10 (IL-10), added exogenously or produced by microglia, to down-regulate microglial functions. This report demonstrates that microglia from the adult human brain produce IL-10 following interferon-gamma/lipopolysaccharide activation. Functionally, recombinant human IL-10 down-regulated basal HLA-DR expression by microglia and inhibited, in a dose-dependent response, the ability of microglia to stimulate CD4+ T-cells in antigen presentation assays. These data, together with recent observations of the inhibition of experimental allergic encephalomyelitis (EAE) following IL-10 administration and reduced CNS infection by Listeria monocytogenes after anti-IL-10 treatment, suggest that IL-10 production by microglia may have important immune-regulatory functions in CNS disease and disease models.

Fc receptor for IgG (FcR) on rat microglia

Receptor for IgG (FcR) was demonstrated on rat microglia in vivo and in vitro by immunohistochemical staining with immune complexes of horseradish peroxidase (HRP) and rabbit IgG anti-HRP. Astrocytes, oligodendrocytes and neurons did not express FcR. Microglia in culture also showed FcR-mediated agglutination and phagocytosis of IgG-sensitized erythrocytes. A radiolabelled cDNA probe for rat FcRIII hybridized with a 1.4-kb RNA band in Northern blots prepared from total RNA from rat brain. FcRIII mRNA-positive cells in rat brain, presumably microglia, were demonstrated by in situ hybridization. FcR participates in the initiation of cytotoxic responses and of phagocytosis by microglia and is therefore likely to be important in mediating immune reactions in the brain.

Clinical immunology of chronic cold agglutinin disease

We studied clinical and immunological characteristics of 15 patients with chronic cold agglutinin disease (CAD). Mean age at disease debut was 68 years for female and 67 years for male patients. The patients had no signs of other autoimmune diseases. All patients had VH4–34 encoded IgM kappa cold agglutinins (CA) in high titre. In five patients IgM increased significantly with advancing disease. Seven patients had reduced concentrations of lymphocytes, largely of CD4 and CD8 T cells. Percentages of NK cells (CD56) and B cells (CD19) were increased in seven and three patients, respectively. In six out of nine patients a clonal expansion of kappa positive B cells was found. Serum C3 was decreased in nine patients and C4 was decreased in 11 patients, six of whom had reduced CH50. Such data indicate that patients with CAD experience a continuous low‐grade complement consumption. Five patients had experienced increased haemolysis during infections. After addition of active complement to patient sera in vitro, six sera showed increased haemolytic activity. Our results indicate that some patients with CAD have a relative deficit of complement in their serum and that an increase of complement production occurs during an acute phase reaction which enhances haemolysis. Our data also indicate that both CA titre and thermal amplitude are important characteristics when predicting complement activation and clinical course in CAD.