3 Results
3.1 Patients with limited disease have higher serum rituximab levels
To date the inter-individual variability of response of lymphoma
patients to rituximab is still not understood. To better understand the
turnover of rituximab, we monitored serum rituximab levels of 14
pediatric patients at day five after one dose of rituximab. The levels
showed high inter-individual variety. Interestingly, there was a
significant difference of the five-day rituximab levels between patients
with limited disease compared to patients with advanced disease
(p = 0.0001, Fig. 1). While patients with limited disease (risk
groups R1 and R2) had a median level of 250 µg/ml rituximab, patients
with advanced disease (risk groups R3 and R4) had a median five-day
rituximab level of 40 µg/ml.
3.2 FcγRI and FcγRII expression on monocytes is altered upon rituximab
treatment
Fcγ receptor (FcγR) mediated binding represents a relevant mechanism of
action of rituximab induced B cell depletion in B-NHL. As linkers
between malignant B cells and effector cells, FcγRs promote B cell
depletion via antibody-dependent cellular cytotoxicity (ADCC),
antibody-dependent phagocytosis (ADP) or
trogocytosis.11 However, the importance of FcγR
mediated B cell depletion in vivo is still unknown. Therefore,
FcγR expression was measured in monocyte subtypes of B-NHL cases (n =
10) before and at day five after rituximab treatment. Monocyte subtypes
can be distinguished by the expression of the glycoprotein CD14 and
FcγRIII/CD16 on their cell surface. Vessel wall patrolling cells are
called non-classical monocytes
(CD14lowCD16+) and migrating cells
are called classical monocytes
(CD14+CD16-).12 A
third subset with a different expression profile was discovered later
and is called intermediate monocytes
(CD14+CD16+).13The subtypes are not only distinct in expression patterns, but also in
function.14 FcγRI expression was significantly
increased after rituximab treatment in classical monocytes (p =
0.002). While FcγRIII expression was not altered, FcγRII expression was
significantly decreased in classical and intermediate monocytes at day
five after treatment (p = 0.006 and p = 0.023, Fig. 2).
FcγR expression was not altered in neutrophils
(CD66b+) and NK cells (CD56+)
(Supporting Information Fig. S1 and S2).
3.3 The expression of S100A9 and S100A12 in monocytes and neutrophils is
increased after rituximab treatment while serum levels decreased
S100A9 and S100A12 are small calcium-binding proteins with
proinflammatory properties that can trigger recruitment and activation
of effector cells during early inflammatory events. They are highly
expressed in the cytosol of neutrophils and monocytes during
inflammation.15,16 To study the effect of rituximab on
the early immune response and specifically on the activation of effector
cells in B-NHL patients during treatment, the expression of S100A9 and
S100A12 in monocyte subtypes was monitored before and at day five after
rituximab treatment as shown by the flow cytometric analysis (n = 10,
Fig.3 A). S100A9 expression was significantly increased in classical,
intermediate and non-classical monocytes after rituximab treatment
(p = 0.005, p = 0.009 and p = 0.031, respectively).
After rituximab exposure, S100A12 showed a significantly higher
expression in classical and intermediate monocytes (p = 0.037 andp = 0.024, respectively), whereby non-classical monocytes showed
a similar tendency, although not significant. In addition, S100A9 and
S100A12 expression in neutrophils was significantly increased in
neutrophils at day five after rituximab treatment (p = 0.023 andp = 0.008, Supporting Information Fig. S3). Conversely, serum
levels of S100A8/A9 and S100A12 significantly decreased after rituximab
treatment (n = 10, Fig. 3B, p = 0.021 and p = 0.012).
3.4 NK cell expression of CD57, perforin and granzyme B is decreased
after rituximab treatment
NK cells are important mediators of ADCC and ADP and thereby highly
contribute to rituximab mediated B cell depletion in patients suffering
from B-NHL.17 However, their role during rituximab
treatment in vivo is not fully understood. Therefore, we analyzed
proteins involved in apoptotic and cytotoxic processes. The expression
of CD57, perforin (PRF1) and Granzyme B (GrzB) was analyzed in NK cells
before and at day five after rituximab treatment (n = 12) in the B-NHL
cohort. Surprisingly, CD57, PRF1 and GrzB expression were significantly
decreased after rituximab treatment in NK cells (p = 0.006;p = 0.045; p = 0.028, Fig. 4).