Pediatric Rheumatology Online Journal
Vol 2, No. 2 2004 (119-133)
http://www.pedrheumonlinejournal.org
Leukocyte activation in the HyperImmunoglobulinemia D and periodic fever
syndrome
Joost Frenkel1, Stephanie
E. Smetsers1, Ger T. Rijkers2, Jacobus F. Gaiser2,
Sander M. Houten3, Hans R.Waterham3, and Wietse Kuis2
Affiliations:
- Departments of General Pediatrics and
- Pediatric Immunology, Wilhelmia Children's
Hospital,
- Laboratory Genetic Metabolic Diseases,
Departments of Pediatrics/Emma Children's Hospital and Clinical Chemistry,
Keywords: Mevalonic acid; fever; hyperimmunoglobulinaemia; immunoglobulin
D; autoinflammatory; inflammation; leukocytes; surface antigens
Contact:
Joost Frenkel MD
Div. of Pediatrics Wilhelmina
Children's Hospital
KE.04.133.1,
e-mail: j.frenkel@wkz.azu.nl
tel: +31 30 2504001
fax: +31 30 2505349
Abstract
Objective:
The Hyper IgD and periodic fever syndrome
and mevalonic aciduria are characterized by recurrent episodes of generalized
inflammation. Both syndromes are caused by a deficiency of mevalonate kinase.
How this inborn error in isoprenoid biosynthesis leads to inflammation and
which cells are involved in this process is as yet unknown.
We investigated whether specific
leukocyte populations are activated during the fever attacks in children with
mevalonate kinase deficiency.
Methods:
Blood samples obtained during and between
fever attacks were analyzed by white-cell and differential counting and by
flow-cytometry. Cells were studied for the expression of CD3, CD4, CD8, CD14,
CD20, CD23, CD64, CD69 and HLA-DR.
Results:
Six patients were studied. During fever,
monocyte numbers rose 3-fold and neutrophil granulocytes 4-fold. These cells
were activated, as reflected by the expression of CD64, which was increased
3-fold on monocytes and 6-fold on granulocytes. There were no such changes in
other leukocyte subsets.
Conclusions:
Activation of monocytes and neutrophil
granulocytes is involved in the fever attacks of the Hyper-IgD and periodic
fever syndrome.
Introduction
The
hyperimmunoglobulinemia D and periodic fever syndrome (HIDS), also known as
Dutch type periodic fever (MIM#260920), is an autosomal recessive disorder
characterized by febrile attacks recurring at more or less regular intervals
and the presence of an elevated serum IgD concentration (>100 IU/ml) (1). Over
170 patients have been diagnosed with the disease worldwide (2).
Clinical features during the febrile attacks include cervical lymphadenopathy,
splenomegaly, hepatomegaly, skin rash, oral ulcers, vomiting, diarrhea,
arthralgias and arthritis. Patients often complain of malaise, chills,
headache, nausea or abdominal pain (3). During
these febrile crises, blood tests reflect an acute inflammatory state with
leukocytosis and elevated acute phase reactants e.g. C-reactive protein.
The underlying genetic defect of the
syndrome is a deficiency of the enzyme mevalonate kinase (MK) due to mutations
in its encoding gene, MVK (4,5)
Mutations in the same gene are responsible for mevalonic aciduria (MA,
MIM#251170), a syndrome with episodic fever, mental retardation and dysmorphic
features (6,7). MK
catalyses the phosphorylation of mevalonic acid into 5-phosphomevalonate, an
early step in the isoprenoid biosynthesis pathway. This route produces
cholesterol which, in turn, is a precursor for steroid hormones, bile acids and
vitamin D. Furthermore, this anabolic route yields a number of non-sterol
isoprenoids. The latter are hydrophobic molecules such as dolichol,
polyisoprene side chains, such as those attached to heme-A and ubiquinone, and
the farnesyl and geranylgeranyl side chains of isoprenylated proteins. Currently,
the chain of events that links this metabolic defect to episodic inflammation
is understood only partly. Indeed, much has been learned about the soluble pro-
and anti-inflammatory mediators involved in HIDS. Patient serum contains high
levels of pro-inflammatory cytokines such as interferon-γ and interleukin (IL)-6 (8,9) during fever attacks. Ex-vivo, isolated
mononuclear cells (MNC) from HIDS patients secrete more IL-1β, IL-6, and
TNF-α than MNC from healthy individuals.
It is as yet not known
which leukocyte subpopulation(s) are involved in the inflammatory activation
that characterizes HIDS. We therefore analyzed leukocyte (sub) populations in
peripheral blood of patients during and between fever attacks.
Our aim was to establish whether
there were quantitative changes in leukocyte subpopulations and whether one or
more of these cell types appeared to be activated in mevalonate kinase
deficiency.
Patients and methods
After ethical review
board approval, our pediatric mevalonate kinase deficiency patients with either
the HIDS or the MA phenotype, were approached to participate in the study. After written informed consent by the
patients' parents, blood was drawn by venipuncture in sterile pyrogen-free
heparinized plastic tubes as well as in 500 μl EDTA- anticoagulated
plastic cups. This was done both when patients were free of symptoms and within
the first 24 hours of a febrile attack. EDTA samples were used for white blood
cell counting and white cell differential counting (Cell-Dyn 4000, Abbott
Diagnostics,
100 μl aliquots of heparinized whole
blood were incubated on ice during 30 minutes with diluted antisera. These
monoclonal antibodies were directly labeled with Fluorescine isothiocyanate
(FITC) or phycoerythrin (PE) and added to a final dilution of 1:10 for anti
CD64-FITC (Immunotech,
Statistical analysis:
Cell counts during and between fever
attacks were compared by the paired Student t-test, a 2-tailed p value of
<0.05 being considered significant. Fluorescence intensity was similarly
analyzed. Values are displayed as mean ± the standard deviation. All analyses
were performed using GraphPad Prism 3.0 software (GraphPad Software Inc.,
Results:
Patients:
Six
mevalonate kinase deficiency patients participated in the study. Five of these
had the HIDS phenotype. These have been described previously as Numbers 119,
132, 135, 137 and 139. One child had the mevalonic aciduria phenotype (10).
In all patients, mevalonate kinase deficiency has been
established and mutations have been identified in both alleles of their
MVK-genes. Four were compound heterozygous for the mutant alleles 1129 G>A
(V377I) and 803 T>C (I268T), and one patient carried the V377I and 59 A>C
(H20P) alleles. The mevalonic aciduria patient had only 0.12% residual
mevalonate kinase activity due to mutations 1000G>A and 421-422insG in his MVK genes (11).
White blood cell and differential counts
were obtained during and between febrile episodes in all 6 patients. In five of
these, the data was obtained during 2 fever episodes and in 2 or 3 non-febrile
intervals. Immunocytochemical analysis was performed at least once during fever
and once between attacks.
White blood cells:
The fever attacks were characterized
by leukocytosis which sometimes was extreme (>40x109/l).
Leukocytes on average rose from 7.7x109/l between attacks to 18.9x109/l
during fever (p=0.001).
Lymphoid cells:
During
fever attacks, the percentage of lymphocytes in the differential count
decreased 4-fold. This was largely due to a rise in myeloid cells. Absolute
lymphocyte numbers showed little decrease (Table 1).
T-lymphocytes:
Absolute T-cell numbers, as
determined by CD3 expression, were similar between and during fever attacks.
There was a slight decrease of T-cells as a percentage of mononuclear cells
during fever (Table 2). However, the absolute T-cell number and the percentage
of T-cells were normal for age (12). CD4 and CD8 subsets were also normal for age
and remained stable during and between attacks (Table 2) as did the CD4 / CD8
ratio.
The proportion of T-cells
expressing CD69, an early activation marker, between attacks (0.4-1.4%) was
comparable to that observed by us in 41 healthy adult controls (1.1-1.4%).
During fever there was a small increase in CD69 expression (Table 2). The
percentage of T-cells that expressed HLA-DR was very similar between and during
fever and well within the normal range (13).
B-lymphocytes:
The
percentage of B-lymphocytes, as determined by CD20 expression, remained normal
between and during fever episodes, as did absolute B-cell numbers (Table 2).
B-cell activation was assessed by the MFI of the Fc-epsilon-Receptor II, CD23,
on CD20 positive cells. B-cell activation between attacks was comparable to
that in healthy adult controls. During fever there was no significant change.
Monocytes:
Monocyte numbers rose
2.8-fold during fever attacks. This was not reflected in the monocyte percentage
in the differential count because of a concomitant rise in granulocytes (Table
1). Activation of monocytes was assessed by the presence on the cell surface of
CD64 (Fc-gamma Receptor I). Monocytes were identified by the expression of the
lipopolysaccharide receptor CD14 (Figure 1a). Activation was quantitatively
expressed as the ratio of MFI of CD64 on CD14 positive cells of patients over
that on CD14 positive cells of healthy adult controls (Table 2). During fever
there was a 2.5-fold rise in CD64 expression on monocytes (p=0.012, Figure 1b).
Granulocytes:
Neutrophil
leukocytosis during attacks was striking (Table 1) with absolute neutrophil
counts rising over 4-fold (p<0.001). Band forms were present in a minority
of patients during fever, but could be as high as 15%. Eosinophil counts
decreased during fever (Table 1).
CD64 expression was measured
on granulocytes, i.e. on cells that had the light scattering characteristics of
granulocytes on flow-cytometry (Figure 1c) and were negative for CD14. The MFI
of CD64 rose 6.5-fold during fever (p<0.01).
These changes in both number
and degree of activation of monocytes and neutrophil granulocytes constituted
the main abnormalities observed in mevalonate kinase deficient patients (Figure
1).
Discussion
Mevalonate kinase deficiency
leads to recurrent bouts of generalized inflammation. The chain of events
linking the metabolic defect to the inflammatory phenotype is incompletely
understood. Somehow, inflammatory effector mechanisms are activated. We aimed
to determine in which cell population this occurred. Several leukocyte subsets
could be expected to be activated in this disorder. B-lymphocytes might be
involved, as suggested by the polyclonal elevation of IgD and IgA, typical of
HIDS (14).
T-lymphocytes had been implicated previously, because of the high serum
concentrations of interferon-gamma during fever attacks (15). Cells
of the monocyte/macrophage lineage were expected to be activated. Inflammatory
mediators typically produced by such cells have been found to be secreted in
increased amounts either in-vivo, ex-vivo or both (16;17). Also,
the raised urinary neopterin excetion is indicative of activation of
mononuclear phagocytes (18).
Finally, neutrophil granulocytes could be involved, since granulocytosis is a
well-known feature of the fever attacks in mevalonate kinase deficiency (19).
Despite the small number of
patients studied, our data indicate that it is mainly the non-specific immune
system that is activated during fever episodes. This finding may very well not
be specific for HIDS, but we did not study leukocyte activation in patients
with other periodic fever syndromes nor, to our knowledge, did others.
We could not detect signs of B-cell
activation. The absolute number of T-cells during fever did not change
significantly, but there was a slight decrease in the percentage of T-cells.
This was largely due to the increase of monocytes within the mononuclear cell
population. There was also no increase in HLA-DR expression. The increase in
CD69 expression on T-lymphocytes, though statistically significant, was very
modest. Its biological significance, therefore, remains uncertain.
In contrast, there was a
4-fold increase in the number of neutrophil granulocytes and a 3-fold rise in
monocyte number. Moreover, these cells were activated as reflected by the
raised expression of CD64.
It can not be excluded that
these phagocytic myeloid cells are activated indirectly by some other cell
population. Blood sampling during attacks took place as soon as fever had
become manifest, so any activation preceding the onset of fever would not have
been detected. It is conceivable that T-lymphocytes are involved in the
initiation of the fever episodes, since these attacks are often triggered by
immunizations or infections and the serum concentration of T-cell derived
cytokines is elevated in HIDS. Also, the activation of non-circulating cells,
such as plasma cells (responsible for IgA ad IgD secretion), sessile
macrophages or dendritic cells, would not have been detected by the present
study.
However, the analogy with
the other hereditary periodic fever syndromes would favor a central role for
granulocytes and monocytes. Like HIDS, these are genetically determined
autoinflammatory diseases, i.e. disorders in which inflammation is prominent
but neither infectious organisms nor auto-reactive lymphocytes or
auto-antibodies are involved (20). In two
of these, Familial Mediterranean Fever (FMF),
the Cold-Induced Auto-Inflammatory syndrome / Muckle-Wells / CINCA
syndrome spectrum, the affected gene is expressed exclusively in granulocytes
and monocytes (21-24). In the
third autoinflammatory disorder for which the gene defect is known, the
autosomal dominant TNF-Receptor Associated Periodic Syndrome, the 55kD
high-affinity receptor for Tumor Necrosis Factor-alpha is mutated (25). This
receptor is expressed on many cell types, but among leukocytes, it is present
predominantly on granulocytes and monocytes. We have observed that the
deficiency of mevalonate kinase worsens during fever (26).
Moreover, further impairment of isoprenoid biosynthesis does augment the
secretion of IL-1β by mononuclear cells upon stimulation via T-cells (27). The
body of data therefore favors a model in which the initiation of an attack may
involve T-cell activation. The consequent activation of mononuclear and,
ultimately, polymorphonuclear phagocytes, however, is not controlled due to the
metabolic defect. These cells, and the soluble mediators they produce, then
give rise to the symptoms patients suffer during their attacks. There are
indications that the periodic fever syndromes result from impaired apoptosis of
phagocytes (28,29).
However, whether impaired apoptosis is instrumental in the increase of
monocytes and neutrophils observed in this study remains to be investigated.
The
findings of activation of mainly phagocytic leukocytes suggest that research on
the role of isoprenoid biosynthesis in the immune system should focus on these
cell types (23).
Acknowledgements
We thank Saskia Mandey and
Noortje Tolenaar for their expert technical and logistical assistance and
Andrea Otten for her assistance in preparing the manuscript.
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Table 1
White blood cell and differential
counts in mevalonate kinase deficiency patients during and between fever
attacks.
|
|
|
non febrile |
febrile |
p* |
|
n |
|
14 |
11 |
|
|
white blood
cells |
x109/l |
7.65 (± 3.20) |
18.93 (± 5.7) |
0.001 |
|
neutrophil
granulocytes** |
x109/l |
3.72 (± 3.03) |
14.95 (±
5.97) |
0.001 |
|
polymorphonuclear
granulocytes |
% |
46.5 (± 20.2) |
78.8 (± 9.1) |
0.003 |
|
band forms |
% |
0 |
2.4 (± 4.4) |
n.s. |
|
eosinophil
granulocytes |
x109/l |
0.12 (± 0.08) |
0.02 (± 0.05) |
0.004 |
|
eosinophil
granulocytes |
% |
1.8 (± 1.3) |
0.2 (± 0.4) |
0.002 |
|
monocytes |
x109/l |
0.38 (± 0.16) |
1.07 (± 0.59) |
0.001 |
|
monocytes |
% |
5.5 (± 2.0) |
5.9 (± 3.2) |
n.s. |
|
lymphocytes |
x109/l |
3.42 (± 1
1.78) |
1.91 (± 0.82) |
0.024 |
|
lymphocytes |
% |
46.1 (± 19.4) |
11.0 (± 6.1) |
0.001 |
|
|
|
|
|
|
|
Values are
means (± standard deviation) |
||||
|
* 2-tailed
paired Student t-test |
||||
|
** sum of polymorphonuclear
granulocytes and band forms |
||||
Table 2
Immunocytochemical analysis of
leukocytes of mevalonate kinase deficiency patients during and between fever
attacks.
|
|
|
non febrile |
febrile |
p* |
|
n |
|
10 |
9 |
|
|
T-lymphocytes |
|
|
|
|
|
CD3 |
x109/l |
2.38 (± 1.02) |
2.09 (± 1.16) |
n.s. |
|
CD3 |
%** |
74.2 (± 4.05) |
61.1 (± 11.1) |
0.008 |
|
CD4 |
% |
39.8 (± 9.7) |
40.8 (± 9.9) |
n.s. |
|
CD8 |
% |
22.4 (± 9.1) |
15.5 (± 6.9) |
n.s. |
|
HLA-DR on CD3+ve
cells |
%*** |
5.2 (± 2.5) |
4.0 (± 1.3) |
n.s. |
|
CD69 on
CD3+ve cells |
%*** |
1.0 (± 0.5) |
2.5 (± 0.9) |
0.008 |
|
B-lymphocytes |
|
|
|
|
|
CD20 |
x109/l |
0.26 (± 0.20) |
0.58 (± 0.45) |
n.s. |
|
CD20 |
% |
8.8 (± 4.2) |
15.2 (± 9.5) |
n.s. |
|
MFI of CD23
on CD20 patients |
|
67.3 (± 32.0) |
50.0 (± 17.9) |
n.s. |
|
MFI of CD23
on CD20 controls |
|
99.6 (± 35.4) |
- |
- |
|
MFI ratio |
|
0.76 (± 0.23) |
0.58 (± 0.39) |
n.s. |
|
myeloid cells |
|
|
|
|
|
MFI of CD64
on patient monocytes |
|
110.7 (±
97.5) |
466.4 (±
213.6) |
0.002 |
|
MFI of CD64
on control monocytes |
|
67.0 (± 58.2) |
- |
- |
|
ratio |
|
1.26 (± 0.59) |
3.28 (± 1.53) |
0.012 |
|
MFI of CD64
on patient granulocytes |
|
12.3 (± 0.9) |
109.7 (±
62.1) |
0.002 |
|
MFI of CD64
on control granulocytes |
|
5.7 (± 2.9) |
- |
- |
|
ratio |
|
1.59 (± 1.10) |
10.4 (± 6.5) |
0.003 |
|
|
|
|
|
|
|
Values are
means (± standard deviation) * 2-tailed,
paired, Student t test |
|
|
||
|
|
|
|||
|
** expressed as
percentage of mononuclear cells |
||||
|
***
percentage of CD3 positive cells |
||||
|
|
||||
|
|
||||
Figure 1
Figure 1: CD64 expression on
granulocytes and monocytes.
Data are from a representative when febrile
and in-between fever attacks.
Panel a) Dot plot of side scatter
(SSC) vs. immunofluorescence with anti CD14-PE. Analyses on monocytes were
performed on cells within the indicated gate.
Panel c) Dot plot of forward scatter
(FSC) vs. side scatter (SSC). Analyses on granulocytes were performed on cells
within the indicated gate.
Panels b) and d) CD64 fluorescence
intensity on monocytes (panel b) and granulocytes (panel d) when febrile (black
histograms) and between fever attacks (white histograms).