Did you know? Calcium helps the body fight infection

Microbial Pathogenesis
Volume 24, Issue 5, May 1998, Pages 309-320

Roles of calcium and annexins in phagocytosis and elimination of an attenuated strain of Mycobacterium tuberculosisin human neutrophils

Meytham Majeed, Nasrin Perskvistf1, Joel D. Ernst, Kristina Orselius and Olle Stendahl Department of Medical Microbiology, Linköping University, Linköping, S-581 85,
Sweden Division of Infectious Diseases and Rosalind Russell Research Laboratory, University of California, San Francisco and San Francisco General Hospital, San Francisco, CA,
U.S.A. Received 2 October 1997; accepted 31 December 1997. Available online 9 April 2002.

The phagocytic function of neutrophils is a crucial element in the host defense against invading microorganisms. We investigated phagocytosis and intracellular killing of an
attenuated strain of Mycobacterium tuberculosis (H37Ra) by human neutrophils focusing on the role of the cytosolic free calcium concentration [Ca2+]I and certain cytosolic
calcium-dependent membrane-binding proteins annexins. Phagocytic uptake did not trigger a calcium rise and occurred independently of different calcium conditions, and in
a serum-dependent manner. Changes in the viability of H37Ra were determined by agar plate colony count and a radiometric assay. Neutrophils showed a capacity to kill ingested mycobacteria and this occurred without a rise in [Ca2+] i. The ability to kill H37Ra [Mycobacterium tuberculosis] decreased in the absence of extracellular calcium and when intra-extracellular calcium was reduced. Immunofluorescence staining revealed that during phagocytosis of H37Ra, annexins III, IV and VI translocated localization of annexin I and V remained unchanged. The translocation of annexin IV occurred even when Ca2+-depleted neutrophils ingested H37Ra in the absence of extracellular calcium. We concluded that neutrophil-mediated killing of mycobacteria is a Ca2+-dependent process. The fact that the association of certain annexins to the membrane vesicle containing H37Ra differ from other phagosomes
suggests a selective regulatory mechanism during phagocytosis of mycobacteria by neutrophils.

Calcium spikes in activated macrophages during Fc receptor-mediated phagocytosis 

Jesse T. Myers and Joel A. Swanson Cellular and Molecular Biology Graduate Program and Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor Correspondence: Joel A. Swanson, Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109-0620. E-mail: jswan@umich.edu

Rises in intracellular-free calcium ([Ca2+]i) have been variously associated with Fc
receptor (FcR)-mediated phagocytosis in macrophages. We show here that
activation of murine bone marrow-derived macrophages increases calcium spiking
after FcR ligation. Ratiometric fluorescence microscopy was used to measure [Ca2+]i
during phagocytosis of immunoglobulin G (IgG)-opsonized erythrocytes. Whereas 13%
of nonactivated macrophages increased [Ca2+]i in the form of one or more spikes, 56% of
those activated with lipopolysaccharides (LPS; 18 h at 100 ng/ml) and interferon- (IFN-
; 100 U/ml) and 73% of macrophages activated with LPS, IFN- , interleukin (IL)-6 (5
ng/ml), and anti-IL-10 IgG (5 μg/ml) spiked calcium during phagocytosis. Calcium
spikes were inhibited by thapsigargin (Tg), indicating that they originated from
endoplasmic reticulum. The fact that activated macrophages showed a more dramatic
response suggested that calcium spikes during phagocytosis mediate or regulate
biochemical mechanisms for microbicidal activities. However, lowering [Ca2+]i with
ethyleneglycol-bis(ß-aminoethylether)-N,N’-tetraacetic acid or inhibiting calcium spikes
with Tg did not inhibit phagosome-lysosome fusion or the generation of reactive oxygen
or nitrogen species. Thus, the increased calcium spiking in activated macrophages was not
directly associated with the mechanism of phagocytosis or the increased antimicrobial
activities of activated macrophages.

Cell Calcium. 1990 Nov-Dec;11(10):655-63.

Oxidase activation in individual neutrophils is dependent on the onset and magnitude of the Ca2+ signal.
Hallett MB, Davies EV, Campbell AK. Department of Surgery, University of Wales College of Medicine, Cardiff, UK.

Using single-cell ratio imaging of Fura-2-loaded neutrophils, we demonstrate that
the heterogeneity and asynchrony of the oxidase response originates from
variability in the timing and magnitude of the cytosolic free Ca2+ signal. The
Ca2+ signals from individual cells could be classified into four types: (a) type 1, a
transient rise in Ca2+ occurring within 6 s; (b) type 2, an oscillating cytosolic free
Ca2+; (c) type 3, a latent Ca2+ transient significantly delayed (21-56 s); and (d)
type 4, no significant Ca2+ rise. These response types accounted for
approximately 41%, 15%, 26% and 18% of the population respectively for
stimulation with 1 microM f-met-leu-phe peptide (n = 27) and 52.5%, 15%,
11.5% and 21% respectively for 0.1 microM f-met-leu-phe peptide (n = 52). The
oxidase in neutrophils in which the cytosolic free Ca2+ concentration rose to
greater than 250 nM always became activated. In the presence of
extracellular Ca2+, cytosolic Ca2+ rose uniformly throughout the cell,
whereas in the absence of extracellular Ca2+, a localized Ca2+ ‘cloud’ was
observed in approximately 30% of cells. A localized activation of the oxidase
accompanied the presence of the Ca2+ ‘cloud’ when the 250 nM Ca2+
threshold was exceeded. The data presented here therefore demonstrate a
tight coupling in individual neutrophils between an elevation in cytosolic free
Ca2+ above a threshold of 250 nM and activation of the oxidase.

PMID: 1965710 [PubMed – indexed for MEDLINE] Science. 1986 Jun 20;232(4757):1554-6.

Calcium modulation activates Epstein-Barr virus genome in latently infected cells.
Faggioni A, Zompetta C, Grimaldi S, Barile G, Frati L, Lazdins J.

In many viral infections the host cell carries the viral genome without producing
viral particles, a phenomenon known as viral latency. The cellular mechanisms
by which viral latency is maintained or viral replication is induced are not
known. The modulation of intracellular calcium concentrations by calcium
ionophores induced Epstein-Barr viral antigens in lymphoblastoid cell lines
that carry the virus. When calcium ionophores were used in conjunction with
direct activators of protein kinase C (12-O-tetradecanoyl phorbol-13-acetate
and a synthetic diacylglycerol), a greater induction of viral antigens was
observed than with either agent alone. Activation of protein kinase C may be
required for the expression of the viral genome.

PMID: 3012779 [PubMed – indexed for MEDLINE] April 17, 2003

How do cells signal and attack foreign matter?
U-M Kellogg Eye Center researcher’s high-speed images show how cells mobilize for immune response
ANN ARBOR, MI – New high-speed imaging techniques are allowing scientists to show how a
single cell mobilizes its resources to activate its immune response, a news research study shows.
Howard R. Petty, Ph.D., professor and biophysicist at the University of Michigan Health System’s Kellogg Eye Center,
has dazzled his colleagues with movies of fluorescent-lit calcium waves that pulse through the cell, issuing an
intracellular call-to-arms to attack the pathogens within. He explains that these high-speed images provide
a level of detail about cell signaling that simply wasn’t possible just a few years ago.

In the April 15 issue of the Proceedings of the National Academy of Sciences, Petty provides more detail on cell
signaling, depicting what he calls the “molecular machinery” underlying the immune response. He has identified a
sequence of amino acids (LTL) that controls the calcium wave pathway and, crucially, the ability
of immune cells to destroy targets. The findings are important because they could eventually lead scientists to design drugs based
on the amino acid motif. “Our clinical goal,” explains Petty, “is to characterize the
immune cell’s signaling function so that we can interrupt it or somehow intervene when it begins to misfire.” The process
has implications for treating autoimmune diseases such as arthritis, multiple sclerosis, and the eye disorder uveitis.

Through images of phagocytosis, the process by which a cell engulfs and then destroys its
target, Petty is able to track the movement of calcium waves as they send signals to key
players in the immune response. The “calcium wave” is a stream of calcium ions coming into the cell, which is detected by the fluorescence emission of a calcium-sensing dye.

As a cell membrane begins to surround its target, two calcium waves begin to circulate. When the target is completely surrounded, one wave traveling
In phagocytosis, a wave traveling around the cell’s perimeter splits in two, with the
second wave encircling the phagosome or sac-like compartment. This second wave
allows the digestive enzymes to enter the phagosome and destroy the target.

When a mutation is introduced, phagocytosis is not completed because the
calcium wave circles the cell and bypasses the phagosome altogether.
around the cell’s perimeter splits in two, with the second wave encircling
the phagosome or sac-like compartment. This second wave allows the
digestive enzymes to enter the phagosome and finally destroy the target.

When Petty introduced a mutation in the gene (FcyRIIA) that controls phagocytosis, he found that
the calcium wave simply circled the cell and bypassed the phagosome altogether. As a result, the
immune cell could engulf, but could not carry out the destruction of its target. This led him to
conclude that the LTL sequence orchestrates the cell signaling process.
The sequence may also have a role in directing other cell activities, for example signaling the
endoplasmic reticulum to form a spindle that connects the phagosome and the outer cell
membrane. “The spindle seems to act as an extension cord that signals the calcium wave into the
phagosome to finish the attack,” suggests Petty.
Petty explains that many of these findings are possible thanks to high-speed imaging techniques
that enable him to merge knowledge of physics with cell and molecular biology. He uses high
sensitivity fluorescence imaging with shutter speeds 600,000 times faster than video frames.
“Before the advent of high-speed imaging, you could not ask many of these questions because
we had no way to see the movement of calcium waves,” he says. “With conventional imaging you
ended up with a blur of calcium.” By contrast, Petty’s images resemble the movement of a comet
across the night sky.

In the study reported in PNAS, Petty used leucocytes as a model for the process. The amino acid
sequence is in the region of the gene FcyRIIA. He is currently studying the same phenomena in
the eye, where phagocytosis disposes of the regularly-shed remnants of photoreceptor cells.
The paper, Signal sequence within FcRIIA controls calcium wave propagation patterns: Apparent
role in phagolysosome fusion, also appears on the PNAS internet site at www.pnas.org.
In addition to Petty, authors on the paper include Randall G. Worth, Moo-Kyung Kim, Andrei L.
Kindzelskii, and Alan D. Schreiber.

Cellular and Molecular Life Sciences (CMLS)
Publisher: Birkhäuser Verlag AG ISSN: 1420-682X
Issue: Volume 58, Number 11/October 2001 Pages: 1727 – 1733

Role of serum components in the binding and phagocytosis of oxidatively damaged erythrocytes by autologous mouse macrophages
K Tanaka A1, Y Usui A1, S Kojo A1
A1 Department of Food Science and Nutrition, Nara Women’s University, Nara
630-8506 (Japan), Fax + 81 742 302459, e-mail: kojo@cc.nara-wu.ac.jp


Abstract. To investigate the role of autologous serum components in the
recognition of damaged cells by macrophages, we examined the binding and
phagocytosis of damage oxidatively damaged red blood cells with Cu2+ and
ascorbate (oxRBCs — oxidatively damaged red blood cells) by autologous resident
mouse peritoneal macrophages. The binding of oxRBCs by macrophages was
independent of the presence of serum. However, phagocytosis by macrophages
increased with serum concentration, and macrophages showed little ingestion of
oxRBCs in a serum-free medium. Macrophages neither bound nor appreciably
ingested native RBCs (before oxidation) in either the absence or presence of
autologous serum. Mouse macrophages ingested significantly more native as
well as oxRBCs in the presence of heat-inactivated fetal calf serum than in the
presence of heat-inactivated mouse serum. Pretreated oxRBCs with normal
serum were rarely ingested by macrophages in a serum-free medium.
Phagocytosis of oxRBCs was significantly inhibited by depletion
of IgG* or calcium from serum, by heat inactivation of
complement, or by antiserum against mouse C3. These results
demonstrate that serum components such as IgG, C3, and
calcium are involved in phagocytosis of oxRBCs by autologous

* IgG : A class of immunoglobulins that include the most common antibodies circulating in the
blood, that facilitate the phagocytic destruction of microorganisms foreign to the body, that bind to
and activate complement, and that are the only immunoglobulins to cross over the placenta.