BCG PROPHYLAXIS INDUCES ACTIVATION OF RECRUITED
NEUTROPHILS
Stefano Ciciliato1, Francesca Vita2, Rita Abbate2, Violetta Borelli2, Laura Toffoli1, Maria
Rosa Soranzo2, Giuliano Zabucchi2, Salvatore Siracusano1
1) Clinica Urologica – Università di Trieste
2) dipartimento di Fisiologia – Università di Trieste
SCOPO DEL LAVORO
Bacillus Calmette-Guerin (BCG) is used to treat high risk superficial bladder cancer, but its
anti-tumor effect remains incompletely defined. Recently a role for polymophonuclear
(PMN) neutrophils has been suggested.
With the aim of more deeply investigating the role of granulocytes, we monitored the
activation state of these cells in the urine of treated patients.
MATERIALI E METODI
Ten patients with pT1G3 TCC of bladder received an 8 week course of BCG therapy.
Pretreatment urine (PRU) and post-treatment urine (POU) samples were collected and
analyzed The cells in the pellet were re-suspended in phosphate buffer saline (PBS) and
counted in counting chambers; differential counts were carried out on cytospin prepared
and stained smears. The numbers of PMN, UC (urothelial cells) was determined
RISULTATI
PRU and POU were evaluated for protein content, number of PMN recruited, elastase
activity, and number of UC.
any significant relationship between the number of
transmigrated neutrophils and the UC detached the morphology of the PMN found in the
urine before the fifth instillation was very similar to that of those PMN found in the urine 23 hours after the fifth instillation with the exception that the former were less numerous and
contained more apoptotic and necrotic cells. The cell populations however have few
secretory granules than blood PMN and contained many of the described neutrophil
extracellular traps (NETs) .PMN isolated after treatment produced superoxide anion (O2)
at a higher levels than that produced by resting blood PMN isolated from the same patient.
The experiments carried out with different PMN preparation revealed that, a PMN
stimulating activity was present in the pooled urine (both POU and PRU) isolated from
BCG-treated patients. In fact blood PMN exposed to patients’ urine, but not urine from
healthy controls, were induced to produce a weak but statistically significant amount of O2
and release their granule contents. Furthermore, after exposure to patients’urine, PMN
from healthy donors acquired a strong cytotoxic activity against T24 cells in culture, while
PMN exposed to urine samples from healthy subjects gave no effect at all. Patients’ urine
or PMN alone had no effect on T24 cells.
DISCUSSIONE
PMN when recruited into the bladder after the BCG immunotherapy are in an activated
state two hours and even up to one week after treatment. Our findings suggest that other
stimuli work with BCG in inducing neutrophil activation We think it more likely that BCG
activate urothelium to produce chemokine’s, which together with other factors induce
neutrophil recruitment.
After this step BCG is eliminated almost completely and PMN begin to reach the bladder
coming from the capillary blood vessels and transmigrating through urothelium. During this
complex migration they are likely activated by large number of cytokines produced by
urothelium
CONCLUSIONI
Our results provide further evidence to support the role of PMN neutrophils in BCG
immunotherapy