V
Vanadate is a pentavalent monomer of vanadium oxide
that can exist either as the meta- or ortho- form depending on the number of oxygen ligands (meta- if
n=3; ortho- if n=4) about the vanadium atom.
Vanadium and the Immune System
3
Vanadium and the Immune System
Mitchell D Cohen
Department of Environmental Medicine
New York University School of Medicine
57 Old Forge Road
Tuxedo, NY 10987
USA
Definition
Although Andres del Rio was first to “discover” vanadium in 1801 (he named it erythronium), he later
came to believe that he had only rediscovered lead
chromate. Credit for its true discovery in 1831 went
to Nils Sefstrom who, using iron ore, was the first to
isolate an oxide of a new metal that he termed vanadium in honor of the Norse goddess of love and
beauty, Vanadis.
Putative Interaction with the Immune System
Putative Non-Immune System Interactions and
Toxicities of Vanadium Agents
While vanadium has been shown to be a mutagen and
a clastogen in numerous mammalian and prokaryote
systems, little is known regarding carcinogenic/mutagenic effects of vanadium agents in humans and animal models; in addition, only a few studies regarding
its teratogenic/embryotoxic effects exist. Following
life-long feeding of rodents with tetravalent vanadium, there was inconclusive evidence for carcinogenicity. This is likely the result of the low level of gastrointestinal uptake, as is the case with many carcinogenic metals that do not display carcinogenic potentials.
In contrast, studies using rodents inhaling V2O5 indicated a dose-related increase in incidence of pulmonary/sinonasal epithelial hyperplasia and metaplasia.
Epidemiological studies noted that acute and/or chronic exposure to moderate-to-high levels of V2O5 or vanadate in dusts/fumes resulted in increased localized
fibrotic foci and lung weights, and an enhanced incidence of lung cancer initiated by other agents.
3
3
Vanadate
3
The combining power of a atom with respect to its
ability to gain, lose, or share electrons in its outer
orbitals/shells.
Chromium and the Immune System
3
3
Valency
Vanadium (V), a group VB transition element, can
exist in multiple valences (0, +2, +3, +4, +5) in both
anionic and cationic forms. Although the tetravalent
and pentavalent forms are the most stable, discrete
ions of each do not exist in nature. Most commonly,
these ions are bound to oxygen as negatively-charged
polymeric oxyanions that readily complex with polarizable ligands such as S or P. In nature, pentavalent
vanadium is most often encountered in the form of
vanadium pentoxide (V2O5), though ferrovanadium,
vanadium carbide, and various forms of vanadates
also exist. Colloidal V2O5 can liberate vanadate (VO3−
and VO4) agents by loss of water, and the resulting
monomeric vanadate ions can be further converted to
higher polymeric forms (Figure 1), akin to how chromate ions link during olation. These conversions, and
therefore the distribution, of vanadium species in solution depend on pH and vanadium concentration. As
a rule, as vanadate unit numbers in the polymer increase, overall toxicity declines; however, even large
polymers like decavanadate can give rise to toxicities.
3
See T cell antigen receptor.
Superantigens
Characteristics
3
Vβ
682
Vanadium and the Immune System
Vanadium and the Immune System. Figure 1 Formation of vanadates and higher polymers from vanadium
pentoxide.
Apparently, vanadium might not act as a direct carcinogen,
but
rather
it
exerts
secondary
( immunosuppressive) toxicities in hosts, allowing
initiated cancers to progress to neoplasms. It should
be noted that certain vanadium compounds have also
been shown to act as anticarcinogens. When given to
rodents bearing Erlich ascites or liver tumors, vanadocene displays cancerostatic activity. In addition, certain vanadium compounds in the form of dietary supplements have been shown to block cancer induction
by other known carcinogenic agents. While the me-
chanisms of the anticarcinogenic activity in these studies are not clear, it should be noted that the effect
observed with vanadocene is not unique; similar results have been obtained using metallocene complexes
with other transition elements.
Putative Immune System Interactions and Immunotoxicities of Vanadium Agents
In immune system cells (as with all cell types), vanadate ions are able to enter the cytoplasm through the
channels utilized by phosphate and chromate anions;
3
Vanadium and the Immune System
3
Exposure also produced pathological alterations in immune system organs (Peyer's patches, thymus, and
spleen). Studies with macrophage cell lines or mice
exposed to NH4VO3 noted decreased surface levels
of Fc-receptors for immunoglobulin binding and diminished production and activity of tumor necrosis
factor(TNF)-α and IL-1α. The latter study also
showed that in the absence of exogenous stimuli, vanadium-exposed macrophages released significantly
greater amounts of inflammatory prostaglandin E2
than did untreated controls.
Studies of host resistance to infection, by Listeria
monocytogenes after acute/subchronic vanadate exposure reported that resident peritoneal and alveolar macrophage function, and consequently cell-mediated immunity, were adversely affected. At the sites of infection, bacterial numbers increased rapidly, but no increase in macrophage or neutrophil numbers occurred.
Macrophages recovered from vanadate-treated mice
displayed decreased capacities to phagocytoze opsonized Listeria or to kill those few organisms ingested.
These defects were thought attributable to vanadiuminduced disturbance in cell superoxide anion formation, glutathione redox cycle activity, and hexosemonophosphate shunt activation, events critical to
maintaining energy for phagocytosis/intracellular killing.
While precise mechanisms underlying the immunomodulatory effects of vanadium are not yet clear, in vivo
and in vitro studies have begun to yield information to
enable hypothetical mechanisms to be proposed. In
macrophages (as well as other cell types) vanadate
ions have been shown to:
* disrupt microtubule and microfilament structural integrity
* induce alterations in local pH due to vanadate polyanion formation
* modify lysosomal enzyme release and activity
* alter secretory vesicle fusion to lysosomes
* disrupt cell protein metabolism at both the level of
synthesis and catabolism
* modulate both the inducibility and magnitude of
reactive oxygen intermediate formation/release.
3
insoluble forms of vanadium enter through pinocytic
uptake. Once in the cell (Figure 2), vanadate is rapidly
acted upon by cellular reductants (such as NAD(P)H,
glutathione, ascorbate, catechols) and converted to tetravalent vanadyl species.
Unlike other toxic metal oxyanions (such as chromate), vanadyl can then either be bound to proteins
or can be readily oxidized back to the vanadate ion. As
it is thought that only pentavalent vanadium can exit
the cell, this represents a means of detoxification.
However, shuttling back and forth between oxidation
states also represents a means for retoxification. Not
only are levels of cellular reductants depleted during
shuttling events, but pentavalent vanadium gives rise
to its own toxic effects, including generation of reactive oxygen species, inhibition of enzymes in nuclear
and cytoplasmic processes, and alterations in the
phosphorylative balance of several proteins secondary to inhibition of cellular phosphatases.
As either vanadate or pentoxide, vanadium has been
shown to alter immunological responses in humans
and experimental animals since the early 1900s. Its
immunosuppressive effects became apparent long
after its initial pharmacological use as an immune enhancer. Workers exposed to atmospheric vanadium
had increased occurrences of coughing spells, tuberculosis, and respiratory tract irritation. Postmortem examinations of these workers revealed extensive lung
damage; the primary cause of death was most often
respiratory failure secondary to bacterial infection.
Later studies demonstrated that acute exposure to
high, and/or chronic exposure to moderate, levels of
vanadium-bearing dust or fumes resulted in higher incidence of several pulmonary diseases, including asthma, rhinitis, pharyngitis, pneumonia, and bronchitis.
Detailed cytologic studies with cells from these exposed workers noted vanadium-induced disturbances
in neutrophil and plasma cell numbers, immunoglobulin production, and lymphocyte mitogenic responsiveness.
Vanadium-induced changes in human immunological
function are reproduced in animal models. Subchronic
and acute exposures of rodents to pentavalent vanadium agents have been shown to alter
* mitogen-induced lymphoproliferation
* alveolar/peritoneal macrophage phagocytosis and
lysosomal enzyme activity or release
* host resistance to bacterial endotoxin (LPS) and
intact microorganisms
* lung immune cell populations
* in situ induction of interferon-γ (IFN-γ) and interleukin(IL)-6 by polyinosinic-polycytidilic acid
* mast cell histamine release.
683
Though these structural/biochemical changes may directly contribute to immunomodulation, they may
have underlying roles in a reduced ability of vanadium-exposed cells to interact with, and respond to, signaling agents during an immune response. Disrupted
endocytic delivery of surface receptor-ligand complexes to lysozomes, subsequent complex dissociation,
and receptor recycling/de novo receptor synthesis, can
diminish the magnitude of macrophage cytokine-induced and/or antigen-induced responses. Along these
lines, studies have indicated that macrophage priming
by T-lymphocyte-derived IFNγ was adversely affected
by vanadium exposure. Activation of cellular protein
kinase A (PKA) or protein kinase C (PKC) and in-
V
684
Vanadium and the Immune System
Vanadium and the Immune System. Figure 2 Metabolism of vanadate after entry into cell and select toxic
manifestations that may result.
creases in intracellular calcium concentrations, events
that can result in a downregulation of IFNγ receptor
(IFNγR) expression, have been observed in macrophages harvested from mice, as well as in J774 murine
macrophage cultures treated with vanadium. In mouse
WEHI-3 macrophages, both the levels of two classes
of surface IFNγR and their binding affinities for IFNγ
were greatly modified by vanadium treatment. In these
cells, IFNγ-inducible responses (including, enhanced
calcium ion influx, MHC class II antigen expression,
and zymosan-inducible reactive oxygen intermediate
formation) were diminished secondary to the changes
in IFNγR expression/binding activity.
Though these indirect mechanisms may clearly be a
means by which decreased expression of surface receptors occurs on vanadium-exposed macrophages, it
is also believed that vanadium may directly modify
receptor proteins themselves (via interactions with
amino acid R-groups in various regions of the proteins). It has also been hypothesized that modified receptor responses may be due to induced changes in
cellular protein phosphorylation/dephosphorylation
states secondary to modifications in the activities of
cell phosphatases. Prolonged phosphorylation of receptor proteins (or cytokine-induced second messengers) might induce false states of cell activation and, as
a result, cytokine receptor modification in the exposed
cells. Similarly, prolonged phosphorylation of proteins
could also lead to bypass of normal signal transduction
pathways and subsequent activation of cytokine DNA
response elements that, in turn, lead to downregulated
cytokine receptor expression/function.
In view of the evidence to indicate that vanadium
compounds are immunomodulating (primarily at the
level of the macrophage; see Figure 3) and the increased concern regarding potential exposures of
human populations, vanadium has now been included
as a US EPA Superfund target inorganic chemical.
Relevance to Humans
Vanadium is one of the more ubiquitous trace metals
in the environment. Since clays and shales can contain
> 300 ppm, coals up to 1% vanadium (by weight), and
petroleum oils 100–1400 ppm depending on site of
recovery, fossil fuel combustion is the most identifiable source for delivery of vanadium-bearing particles
into the atmosphere. Ambient air levels of vanadium
vary from 0.02% by weight in soil-derived aerosols, to
0.02%–0.20% in automobile-derived fumes and
0.54%–0.82% in oil combustion-generated aerosols,
depending on the region under study. Typical rural
vanadium levels are 0.25–75 ng/m3 while urban set-
Vanadium and the Immune System
685
Vanadium and the Immune System. Figure 3 Immunomodulatory events known to occur following in vivo or in
vitro exposure to pentavalent vanadium agents.
tings are usually higher (60–300 ng/m3); on average,
ambient vanadium concentrations in cities are often
several μg per m3. Seasonal variations (winter urban
air vanadium levels are 6-fold greater than summer
levels) arise from increased combustion of vanadium-bearing oils, shales, and coals for heat and electricity. At these levels (≈ 50 ng/m3) and based on
experimental inhalation studies it is estimated that
≈ 1 μg vanadium enters the average adult human
lung each day. Clearance of vanadium from the
lungs depends on solubility of the agent inhaled.
With insoluble V2O5 or more soluble vanadates the
initial clearance is fairly rapid, with ≈ 40% of both
chemical classes cleared within 1 h of intake. However, significant amounts of the cleared material can
enter the systemic circulation and give rise to absorption levels of 50%–85% of an inhaled dose (depending
on agent solubility). After 24 h the two forms diverge
in ability to be cleared, with the insoluble form persisting longer. Thus, total clearance of vanadium is never
achieved, with 1%–3% of the original dose persisting
as long as 65 days or more. As a result, lung vanadium
burden can increase with length of time spent in contaminated environments.
Exposure to vanadium also readily occurs via oral ingestion. Levels of vanadium are higher in freshwater
than in seawater (0.3–200 vs 1–3 μg/l, respectively),
due mostly to saline-induced precipitation of vanadium ions. Municipal water concentrations are usually
< 10 μg/l, so drinking water is not considered an im-
portant source. Food represents the primary source of
noninhaled vanadium intake in both humans and animals. As vanadium levels in most natural foodstuffs
are only several parts per billion, daily human dietary
intake is estimated to be from 0.01–2 mg/day. Unlike
copper, lead, and tin, whose increased presence in
consumable products arises from purposeful supplementation or from product-induced container leaching,
the major contaminating source of foodstuffs by vanadium is soil. After oral intake of vanadium-contaminated water, soil, or foodstuff (or by swallowing vanadium-bearing sputum), absorption from the gastrointestinal tract is low (average 0.1%–1%), irrespective
of the parent compound. There appears to be greater
intestinal uptake of vanadium in younger animals than
in adults, possibly due to greater nonselective permeability of the immature intestinal barrier. Oddly, exposure via noninhalational routes (e.g. per os) can still
give rise to increased lung vanadium burdens and toxicities.
Irrespective of route of entry, vanadium that does enter
circulation is preferentially distributed to the kidney,
liver, blood, and bone. Though each have their own
clearance mechanisms and kinetics, it appears that the
site for long-term vanadium retention is bone. Because
bone acts as a repository for vanadium, possible effects on hematological endpoints, including immune
cell development and function, are great.
Though it has been suggested that vanadium is an essential element for chickens and rats, its essentialness
V
686
Variable Region (V Region)
in humans and most other animals is still not clear. For
now, it appears that only in certain plants (e.g. marine
algae), bacteria (e.g. nitrogen-fixing Azotobacter),
fungi (e.g. Amanita species) and a few lower life
forms (e.g. tunicate ascidians A. nigra, A. ceratodes
and the fan worm Psedopotamilla occelata) does vanadium have some demonstrable function in the host's
biochemical life processes.
Variance, Analysis of
A set of procedures where the total variability from a
set of observations in an experiment is partitioned into
those that account for the systematic or treatment effect, and those that account for chance or random factors.
Statistics in Immunotoxicology
3
References
Variant Antigen Type (VAT)
The highly immunogenic variable surface glycoprotein (VSGs) are at any one point of time of identical
structure in individual trypanosomes and within the
majority of a population in a host resulting in a specific variant antigen type. Only very few individual
parasites express different VATs which are selected
for when the immune response of the host will eliminate all trypanosomes covered by the major VAT.
Trypanosomes, Infection and Immunity
3
1. Cohen MD (1998) Vanadium. In: Zelikoff J, Thomas P
(eds) Immunotoxicology of Environmental and Occupational Metals. Taylor and Francis, London, pp 207–229
2. Cohen MD (2000) Other metals: aluminum, copper,
manganese, selenium, vanadium, and zinc. In: Cohen
MD, Zelikoff J, Schlesinger RB (eds), Pulmonary
Immunotoxicology. Kluwer Academic, Norwel, pp 267–
300
3. Zelikoff J, Cohen MD (1997) Metal immunotoxicology.
In: Massaro EJ (ed) CRC Handbook on Human Toxicology, CRC Press, Boca Raton, pp 811–852
4. ATSDR (1991) Toxicological Profile for Vanadium and
Compounds. US Public Health Service, Atlanta
5. Cohen MD (2004) Pulmonary immunotoxicology of
select metals: Aluminum, arsene, cadmium, chromium,
copper, manganese, nickel, vanadium, and zinc. Journal
of Immunotoxicology I (I):39–69
Vasculitis
3
The segments of immunoglobulin heavy or light
chains that vary in sequence in chains of the same
allotype and isotype, i.e. responsible for antigen specificity.
B Lymphocytes
Rabbit Immune System
An inflammatory reaction of a blood vessel or a lymph
vessel. Vasculitis may occur in many different sites.
Tissue damage starts by complement activation by immune complexes sticking at these sites.
Hypersensitivity Reactions
Systemic Autoimmunity
3
Variable Region (V Region)
3
3
Vasculopathy
Vasoactive Amine
A molecule released from mast cells, basophils, and
platelets that induce contraction of endothelium and
smooth muscle (examples: histamine and 5-hydroxytryptamine).
Serotonin
3
Surface coats (synonym 'glycocalyx') are present in
pathogenic protozoan and helmintic endoparasites.
The body of parasites is covered with variable surface
glycoproteins (VSGs), which are anchored through a
glycosyl phosphatidyl inositol lipid (GPI-anchor).
VSGs are highly immunogenic. Regular changing of
the VSG results in different variant antigen types
(VATs), an immune escape process of the parasite
called antigenic variation.
Trypanosomes, Infection and Immunity
Any disease of blood vessels.
Systemic Autoimmunity
3
Variable Surface Glycoprotein (VSG)
3
Viability, Cell
VDJ
Rearrangement and joining of the V (variable), D (diversity), and J (joining) gene segments is responsible
for the vast potential repertoire of different heavy
chain variable regions in the B cell receptor.
B Lymphocytes
3
VDJ Region
The genetic code for immunoglobulin variable domain
J.
Animal Models of Immunodeficiency
3
Viability, Cell
Andrea Engel
BD Biosciences
Life Science Research
Tullastr. 8–12
D-69126 Heidelberg
Germany
Synonyms
Percent of living cells, live rate, live-death discrimination.
Short Description
Cellular viability indicates the proportion of live cells
in a population. The methods for determination of cell
viability are based on different parameters. These parameters are the membrane integrity, the physiological
status e.g. enzyme activity, othe electrochemical gradient between intact cell compartments or the capacity
of proliferation. A more indirect, exclusive parameter
is the measurement of cell proliferation by labeling of
the dividing DNA molecules in viable cells.
Characteristics
Measurable characteristics of cell viability, are:
* the integrity of the cell membrane
* the physiological status of the cell
* electrochemical gradients
* the capacity of proliferation.
These provide the basis for several types of assays for
cell viability (summarized in Table 1), which can be
monitored by colorimetric detection, fluorescence detection (microscopy or flow cytometry) or radioisometric detection. This chapter summarizes the meth-
687
ods for discrimination of living, damaged, and dead
cells. Methods for monitoring apoptosis are covered in
another section.
Membrane Integrity
Proof of the membrane integrity can be evaluated
using a number of dyes that specifically label dead
or damaged cells. All these probes accumulate in
dead or damaged cells and do not enter viable ones.
The use of fluorescent DNA binding probes such as
propidium iodide (PI), 7-amino-actinomycin D (7AAD) and TO-PRO-3 without thiazol orange in a
flow cytometric method is well established. This method is simple, and the dead and damaged cells are
clearly distinguishable from the viable ones.
7-AAD and TO-PRO-3 allow somewhat more flexibility in combination with fluorochromes other than PI .
These methods are applicable to bacteria, mammalian
cells, protozoa and yeasts (2,4,5,12). Identification of
living cells is afforded by thiazolorange (TO) or several of its derivatives, such as the SYBR probes,
YOYO-1 or TOTO-1 (molecular probes). Combination of ‘live cell probes’ with ‘dead cell probes’ such
as TO or SYBR with PI or 7-AAD thus allows definition of viable, injured and dead cells (2). An example of a flow cytometric analysis is shown in Figure 1.
The use of the TO derivatives YO-PRO-1 and TOPRO-1 has advantages because they do not affect the
proliferative capacity of the viable cells. In addition,
these probes show a higher DNA affinity and are also
valid for the detection of apoptotic cells. An example
for dye combination is the use of ethidium bromide
(EB) with acridine orange (AO). EB penetrates only
dead or injured cells due to the loss of their membrane
integrity and stains these cells red. Viable cells appear
green caused by AO.
For evaluation using fluorescence microscopy, several
different fluorescent probes are available (4). For example, the combination of AO and EB is widespread.
A very fast and uncomplicated means of live–dead
discrimination is afforded using light microscopy.
This direct observation is primarily used before further
cell culture. Morphologic changes can be determined
as the simplest criterion. Trypan blue is a commonly
used dye (6). It is actively excluded by viable cells
with intact cell membranes. Nonviable cells retain the
dye and are stained. Another dye, erythrosin B is used
in a similar way, but is not that widespread in use.
The detection of intracellular enzymes like DNase or
trypsin in the cellular environment can also be used to
indicate the existence of dead cells. Also the penetration of cytoplasmic markers (like antitubulin, anticytokeratin-specific antibodies) can prove the presence
of damaged plasma membranes (4).
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688
Viability, Cell
Viability, Cell. Table 1 Summary of probes and detection systems which can be used for the determination of
viable and non-viable cells
Membrane integrity
Analyte
Cells detected
Detection system
PI, 7-AAD, EB, TO-PRO-3
Nonviable
Fluorescence (e.g.
FCM, IF)
TO, SYBR derivatives, YOYO-1, TOTO-1, YO-PRO- All (viable and Fluorescence (e.g.
1, TO-PRO-1
nonviable)
FCM, IF)
Escape of intracellular enzymes
Nonviable
Colorimetric (e.g.
ELISA)
Penetration of cytoplasmic markers
Nonviable
Fluorescence (e.g. IF,
FCM)
Enzyme activity Fluorescein diacetate, BCECF, calcein AM, dihydroethidium, MTT, XTT, Wst-1, Wst-8
Viable
Fluorescence (e.g.
FCM, ELISA)
Luminescence
Electrochemical Rhodamine 123, DiOC3
gradient
Viable
Fluorescence (e.g.
FCM, IF)
Colorimetric (e.g.
ELISA)
Oxol
Nonviable
Fluorescence (e.g.
FCM, IF)
Colorimetric (e.g.
ELISA)
3
Viable, prolifer- Radioactive (liquid
ating
scintillation counter)
Proliferation
H-thymidine
Bromodeoxyuridine
Viable, prolifer- Fluorescence (e.g.
ating
FCM, IF)
Colorimetric (e.g.
ELISA)
7-AAD, 7-amino-actinomycin D; BCECF, 2',7'-bis-(2-carboxyethyl)-5-(and-6)- carboxyfluorescein; EB, ethidium bromide; ELISA,
enzyme-linked immunosorbent assay; FCM, flow cytometry; IF, immunofluorescence microscopy; PI, propidium iodide; SYBR,
SYBR (molecular probes); TO, thiazol orange; TOTO, TOTO-3 iodide (molecular probes); Wst-1, 4-[3-(4-iodophenyl)-2-(4nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate; Wst-8, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium; YOYO-1, YOYO-1 iodide (molecular probes).
cells is therefore optimized. Carboxyfluorescein and
calcein AM work in the same way. The long retention
time and the small pH sensitivity of calcein AM are
favorable. Dihydroethidium is taken up by viable cells
and cleaved by esterases to an ethidium monomer,
which binds to cellular DNA and causes their red fluorescence. Dead cells remain unstained. Using this dye
the viable intracellular parasites can also be identified by flow cytometry (4). The choice of the optimal
probes depends upon the cell system. Differences both
in uptake efficiency and in the retention time should be
taken into consideration.
Metabolic activity can also be evaluated using different water-soluble tetrazolium salts (e.g. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT); sodium 3´-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic
acid hydrate (XTT); 4-[3-(4-iodophenyl)-2-(4-nitro3
Physiological Status
Viability can be assessed by verification of a specific
cell function, e.g. an enzyme activity. In most cases,
this measurement is also dependent on the membrane
integrity, in so much as it influences the availability of,
or retention of, the probe. Here one uses a lipid-soluble
probe that readily crosses the membrane, and which is
nonfluorescent, e.g. fluorescein diacetate (FDA). The
activity of cellular esterases in viable cells converts
the probe to a highly fluorescent form (in this case,
free fluorescein). Viable cells retain the fluorochrome
and are strongly fluorescent; nonviable ones are dim or
nonfluorescent. FDA is used with bacteria, protozoa,
phytoplankton, plant cells, yeasts and mammalian
cells (4). One variant of this dye, 2',7'-bis-(2-carboxyethyl)-5-(and-6)- carboxyfluorescein (BCECF) (Molecular Probes), is excluded from the cells in an energy-dependent manner and the retention in the viable
3
Viability, Cell
689
actively exclude these by a glycoprotein pump, thus
leading to weaker staining. An additional technical tip:
in the presence of glutathione, cells may have hyperpolarized mitochondria, which causes unspecific staining of dead or damaged cells. The presence of aliphatic side-chains supports the probe uptake and its retention time in the cell. One example is the cyanine probe
DiOC6. It accumulates more strongly in the mitochondria and works in the same way as rhodamine-123. All
the dyes mentioned here could be combined with
DNA labeling probes, which detect nonviable cells
(e.g. PI or EB combined with rhodamine-123).
Lipophilic anionic probes stain nonviable cells. One
example, oxol, has been used for determinations in
bacteria and protozoa (4, 12). The loss of the negative
potential in comparison to the environment leads to the
accumulation of oxol in dead cells. Some caution in
interpretation is required, in light of a publication reporting PI-negative and oxol-positive bacteria (8).
phenyl)-2H-5-tetrazolio]-1,3-benzene
disulfonate
(Wst-1); 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (Wst-8)).
These are cleaved by the metabolic activity of mitochondria into water-soluble colored formazanes. The
experiments are easily and quickly performed in a microtiter plate format. The measurement system is,
however, strongly influenced by the assigned cell
number and the cell culture environment (e.g. pH, dglucose concentration) (7).
Electrochemical Gradients
Viable cells maintain characteristic electrochemical
gradients across their plasma membrane. The maintenance of the pH and other ion gradients is often affected by the loss of viability. Probes typically used to
measure electrochemical gradients are lipophilic and
charged. They thus concentrate in particular subcellular compartments, depending on the relative membrane potential. Rhodamine-123 (a cationic lipophilic
probe) accumulates in mitochondria. Cells with active
mitochondria are stained bright green, but dead or
damaged cells remain unstained because of the loss
of the ion gradient of their mitochondrial membrane.
Rhodamine-123 is used to assess cell viability for bacteria, yeast and mammalian cells (4). When using rhodamine-123 and some other comparable probes, one
should be aware of the fact that certain cell types
Proliferation
Proliferation is another indication of cell viability. The
ability to form colonies in vitro, after plating at low
density, is one of the oldest, but lengthiest procedures.
More often, a direct measurement of new DNA synthesis is used as a criterion of cell growth. Here, analogues of the DNA base deoxythymidine, e.g. radioactive 3H-thymidine or BrdU, are added to the culture
and are incorporated into the DNA in place of thymidine in cells undergoing DNA synthesis (13, 14). The
level of 3H-thymidine incorporation is determined in a
liquid scintillation counter. The incorporation of bromodeoxyuridine (BrdU) is detected with an anti-BrdU
antibody. The incorporation can be visualized either in
a cellular immunoassay (ELISA) or on a single cell
basis by fluorescence microscopy or flow cytometry.
The most appropriate method for measuring cellular
viability depends on the test conditions and the question to be addressed. Many considerations that are
relevant to the choice have been discussed above. In
addition, safety considerations may require that the
cells have to be fixed prior to analysis. The methods
described thus far are not applicable to fixed cells.
Several modifications of the same types of measurement have, however, proven to be suitable for use
when subsequent fixation is required. For example,
ethidium monoacid (EMA) is positively charged and
labels dead cells by DNA intercalation. Exposure of
the samples to visible light causes cross-linking of the
probe with the DNA so that the surplus dye can be
washed out before cell fixation. A combination of PI
and Hoechst is also convenient. PI incubation prior to
fixation labels the dead cells. After ethanol fixation,
cells are counterstained with Hoechst. PI, which is
only present in the dead cells, quenches the Hoechst
fluorescence and labels these cells red. Styryl-751
3
Viability, Cell. Figure 1 A bacterial sample was
stained using the BD Cell Viability Kit (BD Biosciences)
with thiazolorange (TO) and propidium iodide (PI) and
analyzed on a BD FACSCalibur. All cells are TOpositive. Viable cells are only TO-positive; injured ones
TO-positive and weakly PI-positive; dead cells are
positive for both markers (2).
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690
Vimentin
(LDS-751) penetrates into viable and nonviable cells.
Dead and damaged cells are substantially more intensely labeled than living ones. Cross-linking of the
DNA and the dye is not necessary.
8.
Relevance to Humans
9.
Various guidelines and draft guidelines for cell viability testing are available.
* TGA (Therapeutic Goods Administration) Guidelines for sterility testing of therapeutic goods, 2002
* EPA (Environmental Protection Agency) Health effects test guidelines OPPTS 870.5300: Detection of
gene mutations in somatic cells in culture
* EPA (Environmental Protection Agency) Health effects test guidelines
* OPPTS 870.5375; In vitro mammalian cytogenetics
* Draft guidelines on skin corrosivity
* OECD Guideline 431, March 2001
* OECD Guideline 404, June 2001
* Draft guidelines on phototoxicity
* OECD Draft Guideline 432, March 2003 Thanks to
Dr. Pamela Schu-Werner for her assistance with
corrections in expression and spelling in English.
References
1. Alvarez-Barrientos AJ, Cynton R, Nombela C, SanchetPerez M (2000) Applications of flow cytometry to
clinical microbiology. Clin Microbiol Rev 13:167–195
2. Alsharif R, Godfrey W (2002) Bacterial detection and
live/dead discrimination by flow cytometry. Microbial
cytometry application note. BD Biosciences Immunocytometry Systems, San Jose
3. Alsharif R, Tapia M, Godfrey W, Wanalund J, Nagar M
(2002) Bacterial disinfectant efficacy using flow cytometry. Microbial cytometry application note. BD Biosciences Immunocytometry Systems, San Jose
4. Current protocols in flow cytometry, UNIT 9.2 Assesment of cell viability
5. Graham JK (2001) Assessment of sperm quality: a flow
cytometric approach. Anim Reprod Sci 68:239–247
6. Johnson JE (1995) Methods for studying cell death and
viability in primary neuronal cultures. Methods Cellular
Biology 46:243–276
7. Mosmann TR (1983) Rapid colorimetric assay for
cellular growth and survival: application to proliferation
11.
12.
13.
14.
15.
Vimentin
Belongs to the class III intermediate filaments, together with desmin and glial fibrillary acidic protein. Extracellular matrix protein (∼ 54 kDa molecular mass)
produced by fibroblasts in conective tissue with a
function in maintaining the cell shape.
Immunotoxic Agents into the Body, Entry of
Virulence
The degree of pathogenicity of a microorganism as
indicated by invasiveness and mortality.
Streptococcus Infection and Immunity
3
Regulatory Environment
10.
3
The regulation of cellular viability is an important criterion in the evaluation of in vitro and in vivo experiments. The determination of cellular viability and
growth is critical, for example, in the evaluation of
the effect of cytostatics or antibiotics, in drug-screening for the development of therapeutics, in the search
for optimal fermentation conditions for protein secretion or bioproduction, or the evaluation of mutagenic,
carcinogenic and cytotoxic characteristics of different
substances.
and cytotoxicity assays. J Immunol Methods 65 (1–
2):55–63
Nebe-von Caron G, Badley RA (1996) Bacterial
characterization by flow cytometry. In: Al-Rubeal M,
Emery AN, eds. Flow cytometry applications in cell
culture. Marcel Dekker, New York, pp 257–290
Nebe-von Caron G, Stephens PJ, Badley RA (1999)
Bacterial detection and differentiation by cytometry and
fluorescent probes. Proc Royal Microbiol Soc 34:321–
327
Nebe-von Caron G, Stephens PJ, Hewitt CJ, Powell JR,
Badley RA (2000) Analysis of bacterial function by
multi-colour fluorescence flow cytometry and single cell
sorting. J Microbiol Meth 42:97–114
Shapiro HN (2000) Microbial analysis at the single-cell
level: tasks and techniques. J Microbiol Meth 42:3–16
Sims PJ, Waggoner AS, Wang CH, Hoffman JF (1974)
Studies on the mechanism by which cyanine dyes
measure membrane potential in red blood cells and
phosphatidylcholine
vesicles.
Biochemistry
13
(16):3315–3330
Steel GG (1977) In: Growth kinetics of tumours.
Clarendon Press, Oxford
Takagi S et al. (1993) Detection of 5-bromo-2deoxyuridine (BrdUrd) incorporation with monoclonal
anti-BrdUrd antibody after deoxyribonuclease treatment.
Cytometry 14:640
Thornton R, Godfrey W, Gilmour L, Alsharif R (2002)
Evaluation of yeast viability and concentration during
wine fermentation using flow cytometry. Microbial
Cytometry Application Note. BD Biosciences, Immunocytometry Systems, San Jose
Vitamins
Vitamins
René Crevel
Safety & Environmental Assurance Centre
Unilever Colworth
Sharnbrook, Bedford
K44 1LQ UK
UK
Synonyms
691
vitamin E (α-tocopherol), and vitamins K1 and K2
(phylloquinone, farnoquinone). Water-soluble vitamins include those of the B complex: vitamins B1
(thiamine), B2 (riboflavin), B3 (niacin), B6 (pyridoxine), B12 (cyanocobalamin, pantothenic acid, folic
acid), and vitamin C (l-ascorbic acid).
Characteristics
Given their varied and crucial biological roles, vitamins might be expected to modulate the activity of the
micronutrients, essential trace nutrients
Definition
Vitamins constitute a heterogeneous range of micronutrients which are essential to the maintenance of
good health and, indeed, life itself in many cases.
With some exceptions, they cannot be synthesized
by the organism and must therefore be supplied by
the diet. Vitamins are commonly grouped into fat-soluble and water-soluble types. Fat-soluble vitamins include vitamin A (retinol and its derivatives),
vitamins D2 and D3 (ergocalciferol, calciferol),
V
Vitamins. Figure 1 a
Vitamins. Figure 1 b
692
Vitamins
in vitamin A-deficient mice, accompanied by reduced
antibody responses. The RAR is also involved in immune modulation by vitamin A, according to studies
in mouse Th1 cell clones which revealed that retinoic
acid inhibits IFN-γ production through the CD28 costimulatory pathway.
Retinol also acts as an important cofactor in T cell
activation, apparently influencing the G0 to G1 transition and inducing a marked increase of proliferation
of human peripheral blood mononuclear cells
(PBMC). However, this effect requires special culture
conditions to demonstrate, explaining the apparent
contradiction with early findings that appeared to
show no effect in standard lymphocyte proliferation
experiments. The effect depends on the lymphoid
organ from which the cells originate: CD4+ and
CD8+ thymic cells, but only CD4+ peripheral T cells
respond.
The effects of vitamin A extend to the innate immune
system. Vitamin A acetate was found to inhibit multiplication of tubercle bacilli in macrophages in vitro.
Vitamins. Figure 1 c
immune system, and its functional correlates, including inflammation, resistance to infection, and tumor
progression. Studies investigating such effects have,
however, largely concentrated on a few vitamins.
These include vitamin A and retinoids, vitamin D,
vitamin E and vitamin C. Only a few studies, and in
some cases none, have been undertaken into the effects of the other vitamins on the immune system. This
section will summarize postulated activities and,
where identified, mechanisms for each vitamin.
Vitamin A
Vitamin A is involved in the development of virtually
all cells, with actions at a fundamental level (inhibition
of potassium currents, PKC-associated signal transduction). In cells of the immune system, as well as
in others, these actions are mediated through two
types of receptor, the retinoic acid receptor (RAR),
and the retinoid X receptor (RXR). These receptors
belong to a family of nuclear receptors which also
includes the vitamin D receptor (VDR). Vitamin A
and retinoids drive the immune system towards a
T helper type 2 cell (Th2) phenotype, inhibiting the
development of the Th1 phenotype, an activity
mediated through the RXR. Conversely, vitamin A deficiency leads to a Th1 phenotype, manifested by increased interferon-γ secretion and a downregulation of
antigen-presenting cell activity, as well as reduced secretion of interleukin (IL-5). This was well illustrated
by the development of a Th1 response to the parasite
Trichinella spiralis instead of the normal Th2 response
Vitamin D
The active form of Vitamin D is 1α(OH)2D3, which is
produced by cytochrome P450-dependent metabolism
of calciferol and ergocalciferol, and is also known as
calcitriol. It was initially identified through its effect
on calcium metabolism and bone formation, and is not
a vitamin in the true sense, as it can be produced
endogenously by the action of sunlight on the skin.
More recent studies, including the discovery of the
ubiquitous vitamin D receptor (VDR), have revealed
that it plays a much more wide-ranging role, of which
immunomodulatory effects form part.
Vitamin D modulates the function of both lymphocytes and accessory cells, including antigen-presenting
cells, interacting with the cells through the VDR. Early
studies showed that it downregulated activation of
T helper/inducer T cells, but not suppressor T cells
or B cells. Subsequent work found that it also modulated cytokine production in the PBMCs of both man
and experimental animals, and altered monokine/cytokine production in vivo, particularly reducing secretion of tumor necrosis factor TNF-α. More recent findings indicate that the effect on lymphocytes is principally a skewing of the response away from a Th1 type
towards Th2, with IFN-γ, IL-12, and TNF-α being
particularly affected by suppression, the effect reflecting an enhancement of Th2 manifestations rather than
suppression of those of Th1. Key recent findings suggest that the immunomodulatory effects of vitamin D
may be mediated mainly through their influence on
accessory cells, rather than directly on lymphocytes.
Vitamin D inhibits maturation of dendritic cells and
monocytes, resulting in low level expression of key
markers of maturation (such as surface major histo-
Vitamins
compatibility complex (MHC) [class] II [antigens],
CD40, CD25, and IL-12, but is not cytotoxic or cytostatic except at very high doses. Interestingly, IL-4 is
reported to reverse the effect of vitamin D on monocyte differentiation. Vitamin D also enhances antibody- and complement-mediated phagocytosis.
Vitamin E
Vitamin E is a naturally occurring antioxidant, which
is found in several isomeric forms, the most active of
which is α-tocopherol. In vivo, it counteracts free radical damage and is particularly important to immune
cells owing to the higher concentration of polyunsaturated fatty acids (PUFAs) in their membranes.
Vitamin E is postulated to reduce prostaglandin E2
(PGE2) production, possibly through its action on cyclooxygenase 2 (COX-2), the expression of which is
reduced in vitamin E-loaded macrophages from old
mice to the level normally found in the macrophages
of young mice.
Immune cells exposed to higher levels of vitamin E
and tested in vitro secrete greater amounts of the cytokines IL-2, IFN, IFN-γ, while production of IL-6,
IL-1, and TNF-α (proinflammatory cytokines) is reduced. Vitamin E has also been shown to inhibit programmed cell death due to T cell activation, through
inhibition of CD95 ligand expression.
Vitamin K
Vitamin K (phylloquinone) plays a role in blood clotting, controlling the production of several coagulation
factors. The main symptoms of deficiency are the consequences of coagulation defects (e.g. easy bruising,
impaired clotting). The effects of vitamin K on the
immune system have not been systematically examined, and only anecdotal results are available, without
any exploration of mechanisms.
Vitamin B1
Vitamin B1 (thiamine) is closely involved in intracellular metabolism, in particular the glycolytic pathway
and Krebs cycle.
Vitamin B2
Vitamin B2 (riboflavin) acts as an essential coenzyme
in many oxidation-reduction reactions involved in carbohydrate metabolism. It is thus an important component in the maintenance of oxidant status. One of the
signs of deficiency is cutaneous lesions.
Vitamin B3
Vitamin B3 (niacin, nicotinic acid) also acts as a coenzyme in oxidation-reduction reactions, and deficiency results in severe consequences in many organ systems.
693
Vitamin B6
Vitamin B6 (pyridoxine) participates in protein metabolism as the coenzyme in many enzyme systems. It is
also involved in fat metabolism and in energy transformation in several tissues. Direct addition of
vitamin B6 to lymphocyte cultures indicates that it
plays a role in the induction of serine hydroxymethyl
transferase, an enzyme which is induced during mitogenic stimulation. Deficiency at the cellular level
would therefore be expected to reduce any proliferation-dependent responses. One report suggests
vitamin B6 can bind to the CD4 cell surface receptor,
but this has not been further explored.
Vitamin B12
Vitamin B12 was identified through its role in the prevention of pernicious anemia. Deficiency is also associated with neurological impairment. The biochemical
defect appears to be in the conversion of deoxyuridylate to thymidylate, and also implicates folic acid.
Early studies on lymphocytes from individuals with
pernicious anemia showed reduced proliferative activity in response to mitogens.
Vitamin C
Vitamin C (l-ascorbic acid) was first identified for its
anti-scorbutic activity, but numerous studies since
have demonstrated its strong antioxidant activity. It
influences a wide range of processes dependent on
its oxidative-reduction properties. Specifically it is involved in the synthesis of collagen, carnitine and neurotransmitters, as well as in cholesterol metabolism. A
study in which cultured cell lines were preloaded with
dehydroascorbic acid, showed that it inhibited TNF-α
activation of NFκB activation, and concluded that
vitamin C can influence inflammatory, neoplastic,
and apoptotic processes.
Preclinical Relevance
Vitamin A
Most animal studies on the effects of vitamin A on the
immune system have focused on the effects of deficiency, which remains a problem for a significant proportion of the world’s population. These studies have
largely confirmed those mechanistic studies which indicated that deficiency produced a shift to a Th1 phenotype. An experimental model of infection-induced
(Staphylococcus aureus) arthritis thus showed enhanced T cell responses, but not antibody B cell responses in the vitamin A-deficient rats. The disease
itself was longer-lasting and more severe in those
rats, and measures of innate immunity (complement
activity and phagocytosis) were also depressed. In
line with the Th1 bias, reduced antibody responses
to T cell-dependent antigens, but not to T-independent
ones such as LPS were noted in rats deficient in
V
694
Vitamins
vitamin A, together with reduced natural killer cell and
neutrophil activity. As might be expected, vitamin A
deficiency affects secondary antibody responses as
well as primary ones, as illustrated by the reduced
response of human PBMC from tetanus toxoid immune donors in a vitamin A-deficient SCID mouse
model.
In the mouse, deficiency leads to increased production
of IFN-γ and IL-12, while supplementation with
vitamin A led to the development of a Th2 profile
with increased levels of interleukins IL-4, IL-5 and
IL-10. Effect on the number of T cells and B cells
was relatively modest. Other studies revealed that
vitamin A deficiency reduced secondary (IgG) responses more than primary (IgM) ones, because of
impaired clonal expansion of B cells, rather than reduced antibody production per cell.
Large excesses of vitamin A resulted in increased phagocytic activity in Kuppfer cells from rats, as well as
increased prostaglandin E2 and TNF-α secretion from
those cells as well as peripheral blood mononuclear
cells. Mice supplemented with vitamin A produced
delayed-type hypersensitivity responses to Mycobacterium bovis immunization, whereas unsupplemented
mice did not—even though their diet was adequate
in vitamin A.
The effects of vitamin A have also been investigated in
other species. Chicks showed reduced T lymphocyte
proliferative responses with low vitamin A intakes and
enhanced ones with high vitamin A intakes. Vitamin A
supplementation of the diet of Holstein cows after the
end of lactation led to transient increases in
concanavalin A-stimulated lymphocyte proliferation.
However the effects of supplementation post-partum
and during lactation were more difficult to interpret,
with both suppression and stimulation being observed
under different conditions.
Vitamin D
The effects of Vitamin D on immune responses in
experimental animals are consistent with findings
from in vitro experiments, namely reduced immune
activation—much of it attributable to reduced antigen
presentation. Early experiments demonstrated a reduced antibody response to keyhole limpet hemocyanin (KLH), as well an attenuated delayed-type hypersensitivity response to 2,4-dinitrochlorobenzene
(DNCB). Treatment of female mice with dendritic
cells exposed to Vitamin D prolonged the survival of
syngeneic male skin grafts, and reduced the clearance
of injected male splenocytes. Consistent with those
results, lymph nodes from VDR knockout mice were
significantly larger than nodes from wild type mice,
illustrating how vitamin D modulates immune activation.
Supplementation with vitamin D also demonstrates
clinically significant effects, attenuating or completely
suppressing the disease in animal models of allergic
encephalomyelitis (EAE),type 1 diabetes, and transplant rejection.
Vitamin E
Deficiency of vitamin E in various species of animals
(sheep, pigs, dogs, chicken) reduces a range of commonly assessed measures of immune function, including mitogen responses (T and B cells), IL-2 production, natural killer cell (NK) activity, antibody titer,
plaque-forming cell response to sheep red cell immunization, and phagocytosis by neutrophils and, with
some exceptions, macrophages. Vitamin E deficiency
can also increase the virulence of Coxsackievirus B3
in mice, rendering an avirulent strain pathogenic.
However, vitamin E deficiency can sometimes protect—SCID mice with good vitamin E status, but
that were unable to mount an adaptive immune response, succumbed to infection with Plasmodium
yoelli, whereas their vitamin E-deficient counterparts
survived.
Vitamin E supplementation increases or restores various measures of immune responsiveness in aged experimental animals, mainly mice and rats—delayed
type hypersensitivity (DTH) responses, mitogen responsiveness, IL-2 production. Rats fed a diet high
in vitamin E diet showed increased numbers of
CD4+ cells in thymus, and an increased ratio of
CD4+ to CD8+ (helper/inducer to suppressor/cytotoxic
cells).
Functional measures of improved immune function
following vitamin E supplementation have been demonstrated through reductions in mortality from, or increased resistance to, experimental infections in mice,
and increased resistance to pathogens in chicken,
sheep, and pigs. For instance, the influenza virus titers
of mice supplemented with high levels of vitamin E
were reduced to the same levels as those of young
mice. In a murine AIDS model supplementation with
15 times the normal intake of vitamin E restored immune function, including mitogen responsiveness, NK
activity, cytokine secretion pattern, and reduction of
evidence of hyperactivity. The mechanism was postulated as antioxidant activity protecting against programmed cell death and viral replication.
Vitamin K
In early studies, water-soluble derivatives of vitamin K
act as adjuvants for antibody production in mice to a
soluble protein antigen, although they failed to boost
the induction of delayed-type hypersensitivity. Dietary
studies revealed a protective effect of vitamin K deficiency in tuberculosis, although blood levels were not
measured to verify the deficient state. In chicken,
vitamin K deficiency increased the mortality from in-
Vitamins
fection with the parasite Eimeria tenella, and reduced
the dose of parasite required. In rats, vitamin K and
synthetic analogues are reported to increase the immune response, although no details are given.
Vitamin B1
A thiamine-deficient diet had little effect on the resistance of rats to a normally avirulent Corynebacterium
infection, but severe debilitation might have masked
any subtle signs. Another study showed no difference
in survival after Mycobacterium tuberculosis inoculation between a diet deficient in the B-vitamin complex
and a standard laboratory animal diet, while an excess
of the complex decreased survival. However, the data
do not permit a distinction between the individual
components of the B-vitamin complex. In mice and
guinea-pigs, thiamin deficiency induced by injection
of an antagonist, resulted in thymus atrophy and inhibition of T cell-mediated responses.
Vitamin B2
Vitamin B2 enhanced host resistance in mice to a variety of microorganisms, possibly through stimulation
of innate immunity. At levels above those required to
alleviate deficiency, it also protected against endotoxin
and exotoxin shock, as well as infection by Gram-positive and Gram-negative bacteria.
695
deficiency increased the IgE response to the soluble
antigen dinitrophenyl-ovalbumin.
Vitamin B12
Limited data exist on the effect of vitamin B12 on
immune parameters in animals. Deficiency produced
an increased ratio of CD4 to CD8 lymphocytes in
mice, with a higher proportion of the CD4+ cells secreting IL-4 than IFN-γ. Consistent with these observations, serum IgE levels were increased, while IgG
and IgM levels were reduced, as was the C3 component of complement. Similar findings were observed in
rats, although observations were limited to CD4 to
CD8 ratios and IgG, IgM and C3 levels. Very early
host resistance studies in rodents failed to demonstrate
any adverse effects of vitamin B12 deficiency, but this
may have been due to difficulties in inducing sufficiently profound deficiency.
Vitamin C
Few animal models exist of vitamin C deficiency, because guinea-pigs, fruit-eating bats, and primates are
the only species, apart from man, that do not synthesize vitamin C. Furthermore, in studies of deficiency,
effects on the immune system are potentially very difficult to distinguish from the overall adverse effects on
health.
Supplementation of the diets of mice and weanling
pigs with substantial amounts of vitamin did not
alter measures of immune function, such as lymphocyte proliferation, DTH responses, or antibody production.
Vitamin B3
Only one study examined the effect of vitamin B3 on
the immune system. It found enhanced mitogen-driven
proliferation, as well as increased anti-sheep erythrocyte antibody levels, but a reduced DTH response to
the hapten trinitrochlorobenzene.
Relevance to Humans
Vitamin B6
The effects of vitamin B6 on the immune status of
mice and rats are consistent with reported in vitro
effects on proliferative responses. Most of these effects
have been investigated in the context of antagonism of
vitamin B6 activity by the ammonia caramel contaminant 2-acetyl-4(5)-(1,2,3,4-tetrathydroxybutyl)imidazole (THI). Principal findings from the most comprehensive studies include severe lymphopaenia, affecting particularly the T helper/inducer cell population,
with accompanying decreases in mitogen-driven proliferative responses, as well as T-dependent antibody
responses, and host resistance. One study confirms the
effects on thymic lymphocytes, but reports increased
proliferative activity of thymocytes (not splenic lymphocytes as in other studies) consistent with a reduction of the proportion of immature thymocytes.
Vitamin B6 deficiency induced by administration of
an antagonist (4-deoxypyrindine) reduced the inflammatory and antibody responses to Trichinella spiralis
in mice. A contrasting report relates that vitamin B6
Vitamin A
Vitamin A deficiency remains a major public health
problem in many countries, with impaired immune
functioning well documented as one of the major effects, both in clinical trials and epidemiologic observations. Those studies reveal that the effects of vitamin A
deficiency in people are largely consistent with findings in experimental animal and in in vitro studies.
One study demonstrated underlying abnormalities in
the T cell populations of vitamin A-deficient children,
compared to those supplemented with vitamin A who
had with higher CD4 to CD8 (helper/inducer to suppressor/cytotoxic) cell ratios, higher proportions of
CD4 cells, and lower proportions of CD8 45RO
cells. A study in children confirmed a Th1 cytokine
pattern in vitamin A deficiency, accompanied by an
increased number of NK cells. Interestingly, mortality
was not related to vitamin A levels, suggesting that the
benefits of the Th1 shift in this instance (increased
activity against intracellular pathogens, viruses) may
have outweighed the deleterious ones. However, in
general, studies of vitamin A deficiency in HIV-in-
V
696
Vitamins
fected populations have shown that low serum
vitamin A levels are associated with increased mortality, more rapid disease progression, and increased maternal-fetal transmission of disease. Also, because of
the wide-ranging effects of retinol, deficiency can impair immunity by 'non-specific' mechanisms involving
other cell types, such as inadequate repair of damaged
mucosal surfaces, as well as by reducing the activity of
cells of the innate immune system. A low serum
provitamin A and carotenoid level was associated
with an increased risk for heterosexual HIV acquisition
in patients with sexually transmitted diseases, in a
study in India.
In contrast to some of the equivocal effects on infections cleared handled by Th1 mechanisms, vitamin A
deficiency clearly impairs immunity where Th2 responses are critical. Thus, children who suffered
from vitamin A deficiency and received vitamin A
supplements produced increased antibody responses
to tetanus toxoid and measles. Similarly, supplementation with vitamin A reduced malarial febrile episodes,
as well as spleen enlargement and parasite load, in a
population exposed to the malarial parasite, although
there was no consistent effect on the proportion infected or anemia in that population.
Vitamin D
Studies in man have shown that experimental findings
in vitro and in experimental animals have clinical application. Thus HIV-infected patients have reduced levels of 1α(OH)2D3, and their chronic immune activation can be attenuated by administration of this compound. Vitamin D3 analogues are also used in the
treatment of psoriasis.
Epidemiological data also indicate that some autoimmune diseases (insulin-dependent diabetes mellitus,
rheumatoid arthritis) are more common in regions of
vitamin D deficiency, although a causal relationship
remains to be demonstrated.
Vitamin E
Severe deficiency of vitamin E is generally rare, and
has not been described to the same degree as for other
vitamins. Nevertheless, low serum vitamin E levels
have been associated with an increase in oxidative
stress in HIV-infected individuals, and early studies
showed vitamin E supplementation (twice normal intake) reduced AIDS progression, with beneficial effects on several immune parameters.
Aside from AIDS sufferers, many studies of vitamin E
supplementation in elderly humans have been undertaken, largely to test the hypothesis derived from animal studies that vitamin E could retard age-associated
immune function. Measures of immune function such
as DTH, mitogen responses, and antibody response to
vaccines (hepatitis B, tetanus toxoid) were used to
evaluate the effects. The results generally suggest an
improvement in immune function and reduction of inflammation, but are not sufficient to define an appropriate level of supplementation. Some epidemiological
data support the view that vitamin E supplementation
can restore immune function in the elderly or counteract its decline, but many of the studies are confounded
by other factors, such as concurrent intakes of other
vitamins, the health status of participants, and assessment of vitamin E status. Some intervention studies
suggest that while low-dose supplementation boosts
immune responses, very high doses may reduce it.
Vitamin K
No studies were found of the effect of vitamin K on
the immune system in humans.
Vitamin B1
Vitamin B1 (thiamine) intakes above the recommended daily allowance (RDA) are associated with
improved survival of HIV-infected individuals, and
slow progression to AIDS. Vitamin B1 deficiency is
associated with parasitemia, but the studies do not
demonstrate any relationship between immune function and vitamin B1 deficiency. More interestingly, an
intervention study showed that administration of Bcomplex vitamins restored immune function (DTH,
mitogen response) in surgical cancer patients.
Vitamin B2
An epidemiological study failed to identify a relationship between vitamin B2 (riboflavin) status and parasitemia with Plasmodium falciparum.
Vitamin B3
Several studies have considered the effects of
vitamin B3 on human measures of immunocompetence, but always in combination with other vitamins.
Thus supplementation reduced re-infection with some
parasites in one study, while in another it improved
weight gain in HIV-infected pregnant women. However, these outcomes only suggest a beneficial effect
on immunity, in the absence of measures of immune
function. In another study, niacin intakes were positively correlated with IgG levels in endurance-trained
athletes. Interestingly, supplementation with very large
doses of B vitamins, including niacin, in an elderly
population was associated with reduced circulating
lymphocyte counts, and no evidence of improved immune function.
Vitamin B6
Human studies indicate that vitamin B6 affects lymphocyte maturation and differentiation, with reduced
DTH and antibody responses. Thus, in HIV-infected
individuals at an early stage of infection, reduced
Vitamins
vitamin B6 levels were associated with reduced immune function, manifested by decreased mitogen responsiveness and NK cell activity. No relationship was
found with lymphocyte population profiles or serum
immunoglobulin levels. On the other hand, evidence
does not indicate that supplementation well above normal requirements benefits immune responses.
Vitamin B12
Clinical or epidemiological studies on the effects of
vitamin B12 on the immune system are limited. Consistent with the findings in rats and mice, a small
human study reported increased CD4 to CD8 ratios
against a reduction in total lymphocyte numbers, together with reduced NK cell activity. However serum
immunoglobulin levels were unchanged, as were mitogen-driven proliferative responses. Vitamin B12 administration restored the altered parameters. In a prospective cohort study, HIV-infected individuals with
low serum vitamin B12 concentrations developed
AIDS faster than those with adequate serum levels.
Another study found an association between low
serum levels of vitamin B12 and infection with Helicobacter pylori in healthy individuals.
Vitamin C
Because of the severity of profound vitamin C deficiency, studies have been limited to a few investigations of the effects of moderate deficiency. Lymphocyte proliferation responses were not affected,
although reductions in DTH responses, measured by
skin testing, were observed.
Supplementation with vitamin C has been studied
much more extensively, particularly in relation to the
hypothesis of its beneficial activity against the common cold. Taken together these studies indicate that
vitamin C supplementation does not reduce incidence,
but reduces the severity and duration of the common
697
cold. No mechanism has been identified to date, and
there remain questions over the most appropriate dose.
The studies also show that vitamin C may also protect
against lower respiratory tract infections.
Other studies have investigated whether vitamin C
could protect against the increased susceptibility to
infection following intense exertion. Results are inconclusive. Some studies show a reduction in the incidence of upper respiratory tract infections (URTI)
after racing, but a recent very carefully controlled
study concluded that vitamin C did not alter postrace manifestations of oxidative stress or immunity.
Incidence of post-race URTI was not reported, which
may indicate that any beneficial effect of vitamin C
may be mediated by non-immune mechanisms.
Another study of supplementation revealed a transient
increase in NK cell activity and a reduction in proportion of cells undergoing apoptosis.
Regulatory Environment
There is no regulatory application of the effects of
vitamins on the immune system.
References
1. Calder PC, Kew S (2002) The immune system: a target
for functional foods? Br J Nutr 88 [Suppl 2]:S165–177
2. Erickson KL, Medina EA, Hubbard NE (2000) Micronutrients and innate immunity. J Infect Dis 182 [Suppl 1]:
S5–10
3. Han SN, Meydani SN (2000) Antioxidants, cytokines,
and influenza infection in aged mice and elderly humans.
J Infect Dis 182 [Suppl 1]:S74–80
4. Lin R, White JH (2004) The pleiotropic actions of
vitamin D. Bioessays 26:21–28
5. Meydani SN, Beharka AA (2001) Vitamin E and immune
response in the aged. Bibl Nutr Diet 55:148–158
6. Stephensen CB (2001) Vitamin A, infection and immune
function. Ann Rev Nutr 21:167–192
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