Medical Research Society
The results of this study suggest that although CO1
generation after NaHCO. therapy is inevitable, the
change in intracellular pH is often small and
transient. Limitation of CO, generation (eg slow
addition, increased ventilation) should prevent adverse
intracellular pH changes in the myocardium.
118
THE EFFECT OF BICARBONATE ON THE REGULATION
OF INTRACELLULAR pH BY HUMAN LEUCOCYTES
DJA GOLDSMITH and PJ HILTON
Renal Research Laboratory, St Thomas' Hospital,
London SE1 7EH
Bicarbonate is the most significant intracellular
buffer above pH 7. Many cells have important pH
regulatory mechanisms that are bicarbonate-dependent.
For these reasons it is appreciated that the use of
bicarbonate-free systems (eg Hepes buffer) in
intracellular pH studies can be associated with
misleading results. To elaborate the role of
bicarbonate in human leucocyte pH regulation, we
compared the resting intracellular pH, buffering power,
recovery from intracellular acidosis and the pH
response to a phorbol ester (PMA) of leucocytes in
either Earle's buffer (bicarbonate 24 mmol/l) or Hepes.
Leucocytes were prepared by dextran sedimentation
and osmotic lysis of contaminating red blood cells.
Intracellular pH was measured using suspensions of
leucocytes loaded with BCECF in a fluorimeter.
Intracellular alkalinisation was achieved, and
buffering power derived, by exposure to aliquots of
ammonium chloride. Ammonium chloride prepulse was used
to acidify cells. Intracellular pH recovery was
dependent on the presence of extracellular sodium.
Intracellular pH was higher and buffering power
lower in cells incubated in Hepes compared to cells in
bicarbonate (7.37 2 0.04 vs 7.24
0.02 and 12.2 2 2.1
vs 32.4 2 2.3 mmol/l/pH, n=10, p < 0 . 0 1 ) . The rate of
recovery from intracellular acidosis, and the buffering
power of the cells in acid conditions, were unaffected
by the buffer composition (Hepes: 0.74 pH/min and 42.5
-+ 4.2 mmol/l/pH vs Bicarbonate: 0.78 pH/min and 39.7 2
3.6 mmol/l/pH, n=10, PENS). At resting p~ the cells
were exposed to 5 nmol/l PMA; in Hepes-buffered cells
after 1 5 mins the intracellular pH had increased by
0.42 2 0.08, compared to 0.25 2 0.04 for cells in
bicarbonate (n=8, ~ ~ 0 . 0 5 ) .
In conclusion the presence of bicarbonate profoundly
affects the pH regulation of human leucocytes
119 PATHOGENETIC MECHANISMS IN VASCULITIS, I: ANTINEUTROPHIL CYTOPLASM ANTIBODY (ANCA) STIMULATES UNPRIMED NEUTROPHILS
MT KEOGAN, VLM ESNAULT*, AJ GREEN*, CM LOCKWOOD* AND
DL BROWN
33P
ments produced a significantly increased CL response
which was slow and sustained. This was inhibitable in
each case by azide and salicylhydroxamic acid, but unaffected by superoxide dismutase, suggesting that the
CL resulted from myeloperoxidase (MPO) activity.
The chemotactic response to f-Met-Leu-Phe,a bacterial
chemotactic peptide, but not zymosan activated serum,
was also increased by five of the six ANCA Fab2, while
random motility was not affected. Phagocytosis was
similarly unaffected.
These data demonstrate that ANCA stimulates reactive
oxygen generation by MPO in unprimed normal
neutrophils. The increased chemotactic response to
fMLP may also increase neutrophil accumulation. These
actions may contrGbute to tissue damage in systemic
vasculitis.
120 PATHOGENETIC MECHANISMS IN VASCULITIS, I1 :ANTINEUTROPHIL CYTOPLASM ANTIBODIES (ANCA) ACTIVATE PROTEIN
KINASE C IN HUMAN NEUTROPHILS AND HL-60 CELLS
T FUJIMOTO, CM LOCKWOOD
Department of Medicine, School of Clinical Medicine,
University of Cambridge
We investigated whether ANCA have a pathological effect
on signal transduction mediated by protein kinase C
(PKC): PKC appears to play an integral role in signal
transduction which is an essential step in neutrophil
activation. F(ab')2 fragments were examined from 7 ANCA
positive patients with Wegener's granulomatosis or
microscopic polyarteritis. Two anti-glomerular basement
and 4 normal human IgG
membrane (GBM) antibody F(ab')2
F(ab')2 preparations served as controls. Purified human
neutrophils and human promyelocytic leukemic cell line
(HL-60) cells were treated with ANCA F(ab'I2, normal
F(ab')2, anti-GBM F(ab')2, or phorbol myristate acetate
(PMA). The subcellular distribution of PKC activity
(%PKC) was determined after 60 min. After incubation
with ANCA F(ab')2, there was a significant PKC translocation to the membrane fraction : % PKC activity in
the membrane fraction was 47% (n=9; pt0.05 vs normal
F(ab')Z)
in neutrophils, 28% (n=6; pt0.05) in undifferentiated HL-60 cells and 18% (n=4; ptO.1) in differentiated HL-60 cells. In contrast, PKC was found to
be localised mainly in the cytosol in neutrophils or
HL-60 cells treated with normal F(ab')2:,% PKC activity
in the membrane fraction was 12% (n=6) in neutrophils,
15% (n=6) in undifferentiated HL-60 cells and 4% (n=4)
in differentiated HL-60 cells. Anti-GBM F(ab')2 had the
same effect a s normal F(ab')2: % PKC activity in the
membrane fraction was 5 % (n=4) in neutrophils. Exposure
of neutrophils or HL-60 cells to PMA resulted in a
marked PKC translocation.
These studies indicate that ANCA may modulate
neutrophil activation through translocation of PKC, and
it is therefore possible that direct effects of ANCA on
circulating neut.rophils are important in the
pathophysiology of vasculitis.
Department of Clinical Immunology, Addenbrooke's
Hospital, Cambridge and *Department of Medicine,
University of Cambridge, England
ANCA is a marker for systemic vasculitis for which a
pathogenetic role has been postulated. Neutrophil accumulation appears to play a central role in the blood
vessel wall destruction and granuloma formation in
these conditions. We have therefore investigated the
ability of ANCA to stimulate quiescent unprimed
neutrophils.
Fab2 fragments were prepared from six patients with
ANCA positive systemic vasculitis (3 C-ANCA, 3 P-ANCA)
and two normal controls. Leucocytes were isolated from
normal donors under pyrogen-free conditions and the
chemi-luminescence (CL) response to each of the Fab2
fragments was examined in the presence of luminol, a
photoamplifier. Five of the six ANCA positive frag-
121 PATHOGENETIC MECHANISMS IN VASCULITIS, 111: IgG
SUBCLASS DISTRIBUTION OF ANTI-UYELOPEROXIDASE ANTIBODIES IN SYSTEMIC VASCULITIS
VLM ESNAULT. DRW JAYNE, CE RAWLINSON, AP WEETMAN and
CM LOCKWOOD
Department of Medicine, School of clinical .Medicine,
University of Cambridge, England
Circulating IgG autoantibodies to myeloperoxidase are
associated with renal vasculitis and have been implicated in its pathogenesis. However, raised levels of
these autoantibodies may persist during clinical