Priscilla A. Campbell

Markers of mouse macrophage development detected by monoclonal antibodies

In this review, we present and discuss a selected panel of antibody-defined markers expressed during different stages of mouse macrophage development. We distinguish four categories of markers, which are characteristic of: (1) macrophage precursors and immature macrophages (ER-MP12, ER-MP20, ER-MP54, ER-MP58); (2) mature macrophages in general (F4/80, BM8, Mac-1, Mac-2, ER-BMDM1); (3) macrophage subsets (ER-HR3, ER-MP23, ER-TR9, Forssman antigen, MOMA-1, MOMA-2, Monts-4, SER-4), and (4) IFN-gamma-stimulated macrophages (H-2Ia, LFA-1, ICAM-1, 158.2, MBR-2, TM-2, TM-4, and TM-5). It should be noted that many of the markers in this last category are inducible by other stimuli as well. The rigid classification of markers into four separate groups should be regarded as a digitalization of a continuum, thus inevitably implicating a simplification of the complex phenotypic changes that occur during mononuclear phagocyte development. Nevertheless, the current selection of antibodies against markers for different developmental stages of macrophages constitutes an important tool for characterization of mouse macrophages which participate in various biological processes.

Reexamination of the blood lymphocyte transformation test in the diagnosis of chronic beryllium disease

The T cell response to beryllium, measured in bronchoalveolar lavage by the lymphocyte transformation test (LTT), is a critical diagnostic test for discriminating between chronic beryllium disease (CBD) and other granulomatous diseases. We examined the sensitivity, reproducibility, and methods of a less invasive, peripheral blood LTT in 17 patients with CBD and in 18 beryllium-exposed control subjects. Ninety-four percent of CBD cases (16/17) had abnormal blood LTT results, and all 18 beryllium-exposed control subjects had normal blood LTT results. Split samples for 10 beryllium disease cases and eight control subjects demonstrated that the blood LTT was reproducible between two separate laboratories. The LTT was equally sensitive with 10% and 20% serum in the culture medium. We conclude that an abnormal blood LTT can be used to diagnose CBD in patients with compatible lung pathology.

Screening blood test identifies subclinical beryllium disease.

We evaluated the beryllium-specific lymphocyte transformation test as a workplace screening tool for early beryllium disease. Fifty-one of 58 workers with current beryllium exposure completed questionnaires and gave blood specimens for the lymphocyte transformation test. Six workers (11.8%) had elevated test results ranging from 5.7-fold to 16.7-fold stimulation. Of five who underwent clinical evaluation, four had beryllium disease, demonstrated by granulomata on transbronchial lung biopsy and elevated lymphocyte transformation tests by bronchoalveolar lavage cells (ranging from 18.6-fold to 44.3-fold stimulation). Our data show that (1) minimally symptomatic cases of beryllium disease can be identified by this peripheral blood test, (2) not all individuals with a positive peripheral blood lymphocyte transformation test have beryllium disease at the time of their initial evaluation, and (3) lymphocyte transformation test reproducibility is good, justifying further evaluation of this test for screening. We conclude that the peripheral blood lymphocyte transformation test may prove useful in preventing clinical chronic beryllium disease by early diagnosis in a subclinical phase.

Macrophage phagocytosis: use of fluorescence microscopy to distinguish between extracellular and intracellular bacteria.

One of the challenges of phagocytosis research is to differentiate bacteria adherent to a host cell from bacteria which the cell has internalized. To address this question, various techniques such as fluorescence microscopy, electron microscopy, and flow cytometry have been used. We have adapted a flow cytometric method (Fattorossi et al., 1989) to use fluorescence microscopy for studying phagocytosis of fluorescein-labeled Listeria by inflammatory mouse peritoneal macrophages. In this assay, ethidium bromide is used as a quenching agent and is added to cells after they have phagocytosed labeled bacteria. Ethidium bromide causes extracellular FITC-labeled Listeria to fluoresce red-orange, whereas intracellular bacteria are not exposed to the dye and remain green. This process allows distinction between intracellular and extracellular bacteria by simultaneous visualization of both populations.

Bone marrow cellular composition in Listeria monocytogenes infected mice detected using ER-MP12 and ER-MP20 antibodies: a flow cytometric alternative to differential counting

Detailed assessment of bone marrow cellular composition is essential in the evaluation of various experimental in vivo systems, such as expression of transgenes, null mutations and stimulation of host defence in infection. Traditional morphological analysis of mouse bone marrow is laborious, requires specific cytological expertise, and is somewhat subjective. As an alternative, we have examined whether double labelling of bone marrow with the anti-precursor monoclonal antibodies ER-MP12 and ER-MP20 could be used for differential analysis by flow cytometry, as these antibodies define six relatively homogeneous cell populations in mouse bone marrow. Following a sublethal infection of mice with Listeria monocytogenes, we monitored changes in cellular composition of the bone marrow at various time points in three ways: differential morphological count; single-color flow cytometric analysis using markers for the myeloid, erythroid and lymphoid lineages; and double labelling with ER-MP12 and ER-MP20. As expected, the bone marrow composition changed dramatically during infection, leading to an increase of myeloid cells which peaked after 1 week of infection. Data determined by ER-MP12/20 flow cytometric analysis appeared to be in close agreement with both morphology and lineage marker analysis. In addition, ER-MP12/20 analysis provided more detailed information with regards to the presence of early myeloid precursors compared to lineage marker analysis. These data show that flow cytometric analysis of bone marrow using ER-MP12 and ER-MP20 monoclonal antibodies provides a relatively simple, rapid and objective assay when evaluating cellular composition in the bone marrow of the mouse.

Interleukin-4 is chemotactic for mouse macrophages

An important component of the cell-mediated immune response often is the migration of macrophages to the site of immune activity. Although much evidence suggests that macrophage migration is regulated by antigen-specific T cells, the influence of T cell-derived cytokines on macrophage chemotaxis has not been well studied. Here we present evidence that interleukin-4 (IL-4), a cytokine derived from T helper 2 (Th 2) cells, is chemotactic for mouse peritoneal macrophages. In an in vitro chemotaxis assay using Boyden chambers, recombinant IL-4 was chemotactic for mouse peritoneal exudate macrophages. This response was inhibited in a dose-dependent manner by the anti-IL-4 antibody, 11B11. As shown here and previously, interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), cytokines derived from T helper 1 cells, are not chemotactic for mouse macrophages.

Are inflammatory phagocytes responsible for resistance to facultative intracellular bacteria

At least 38 deaths from listeriosis in the United States in recent months have drawn attention to how little we know about resistance to facultative intracellular bacteria. Much more is known about resistance to obligate intracellular parasites. Macrophages, activated by T cell-derived macrophage activating factors (MAF), are able to kill obligate intracellular parasites and tumor cells(1-10) including Leishmania, certain trypanosomes, Toxoplasma, the obligate intracellular bacterium Rickettsia, and perhaps bacteria such as mycobacteria and Legionella. However, macrophages, stimulated by MAF, may not be the only host cells which can defend against infection by facultative intracellular bacteria such as Salmonella typhimurium or Listeria monocytogenes. Six different observations made by Priscilla Campbell and colleagues, and by others, suggest that it is not the so-called activated macrophage which is primarily responsible for resistance against facultative intracellular bacteria. Rather, she proposes that an early inflammatory cell recently recruited in response to an inflammatory stimulus - a cell whose presence seems to be under the control of immunologically-specific T cells - plays a critical role in resistance to infection by these organisms.

Some macrophages kill Listeria monocytogenes while others do not

Summary: It is not known why some macrophages can kill certain microbes, such as the facultative intracellular bacterium Listeria monocytogenes (L. monocytogenes), while other macrophages cannot. Perhaps listericidal activity is a property of macrophages at specific stages of differentiation; maybe the ability to kill this bacterium is regulated by the microenvironment of the cell; or it is possible that other regulatory forces are important. We describe here three characteristics that distinguish macrophages which can kill I. monocytogenes from those which cannot. First, listericidal macrophages must have neither too much nor too little intracellular iron ‐ they must have an intermediate amount. Second, the receptor a macrophage uses to phagocytose L. monocytogenes seems to influence the intracellular fate of this bacterium. And third, macrophages which have cell‐surface interleukin‐10 (IL‐10), a known down regulator of macrophage function, can‐not kill L. monocytogenes. These trails of macrophages and their effects on listericidal activity are reviewed here, and the possibility that these properties might interact to control macrophage bactericidal activity is discussed.

Beryllium-stimulated production of tumor necrosis factor-α by a mouse hybrid macrophage cell line

Chronic beryllium disease (CBD) results from exposure to the light-weight metal beryllium (Be). In vitro stimulation of bronchoalveolar lavage cells from CBD subjects causes the production of high levels of TNF-alpha, IFN-gamma and IL-6. We tested the hypothesis that Be-stimulation might induce the production of TNF-alpha by macrophage cell lines. We observed that H36.12j cells (12j), a mouse hybrid macrophage cell line, but not other mouse and human macrophage cell lines, produced TNF-alpha upon Be-stimulation. The response was maximal at 100 microM BeSO4 and did not occur when 12j cells were stimulated with either aluminum sulfate or cobalt sulfate. Beryllium-stimulated the production of 725+/-25 pg/ml (mean +/- SEM) TNF-alpha protein by 12j cells as measured by ELISA of culture supernatants after 24 h. As measured by RT-PCR, Be-stimulated 12j cell TNF-alpha protein production was accompanied by an increased intracellular TNF-alpha mRNA at 3 and 24 h. The addition of 10U or 100U of rMu-IFN-gamma to Be-stimulated 12j cells further increased TNF-alpha production 1.5-4 fold (1.6+/-0.1 ng/ml) respectively. Bacterial lipopolysaccharide (LPS, 1 microg/ml) stimulated production of TNF-alpha in 12j culture supernatants after 6 h (515+/-151 pg/ml). This early versus late TNF-alpha production suggests that LPS and Be both stimulate 12j cell TNF-alpha synthesis, but through different pathways. We report for the first time, the direct effects of Be stimulation on the ability of 12j cells to produce TNF-alpha. The 12j cell line, contrasted with other macrophage hybrids that do not respond to Be-stimulation, may provide a useful tool to evaluate the mechanisms by which Be stimulates macrophage cytokine production, and by which T cell derived IFN-gamma amplifies TNF-alpha production in granulomatous diseases.

Mitogenic Effect of Beryllium Sulfate on Mouse B Lymphocytes but Not T Lymphocytes in vitro

Beryllium metal and its salts can produce disease in man and in animal models. Beryllium disease is thought to involve cell-mediated immunity and an antigen-dependent response by beryllium-specific T cells. Beryllium salts have been shown to stimulate lymphocyte proliferation and release of lymphokines, and to induce granuloma formation and delayed-type hypersensitivity reactions in mice, guinea pigs and man. The studies described here were designed to test the hypothesis that a second lymphocyte population, B cells, may be responding nonspecifically to beryllium. Different populations of BDF1 mouse lymphocytes were cultured in the presence of varying concentrations of beryllium sulfate (BeSO4), and the increase in 125-iodouracildeoxyriboside uptake after 72 h in culture was determined. The data show that BeSO4 is weakly mitogenic for normal mouse spleen cells. Furthermore, BeSO4 is mitogenic for normal and nude mouse spleen B cells and not for spleen T cells or thymocytes in vitro. These findings suggest that BeSO4 can stimulate B cells nonspecifically, and support the hypothesis that polyclonal activation of B cells by beryllium may occur.