Prolonged Deleterious Influences Of Chemotherapeutic Agent CPT-11 On Resident Peritoneal Macrophages And B1 Cells

Aim:CPT-11?Irinotecan?is a first-line chemotherapeutic agent for the treatment of colorectal cancer in clinic.Both peritoneal macrophages and B1 cells are among the most important types of tissue-resident innate immune cells in the peritoneal cavity.By interacting with B1 cells,the resident peritoneal macrophages play critical roles in maintenance of gastrointestinal homeostasis.Previous studies including ours have demonstrated that CPT-11 is,however,toxic to the intestinal epithelium and resident peritoneal macrophages.It remains therefore elusive whether these peritoneal innate immune cells could be rebuilt spontaneously or artificially after being impaired by CPT-11 administration.To this end,four experiments were to performed:First,to detect the effect of CPT-11 by intraperitoneal injection on the phenotypes and numbers of intraperitoneal innate immune cells?including peritoneal macrophages and B cells?;Second,syngeneic peritoneal cells and bone marrow cells were transferred into the mice having intraperitoneally treated with CPT-11 7 days before,and the phenotypes and numbers of peritoneal macrophages and B cells in these mice were detected;Thirdly,CPT-11 was administered orally,and the influence of CPT-11treatment on the phenotypes and numbers of peritoneal macrophages and B cells was tested;Fourth,the effect of CPT-11 by intraperitoneal administration on the survival rate of mice against Escherichia coli infection?immune function?were observed.Methods:1.Animal experiment:After one week of acclimatization,C57BL/6?6-8 weeks of age?mice were randomly divided into three groups,one was administered intraperitoneally?i.p.?with vehicle,one was treated with CPT-11?200 mg/kg body weight?and the other was untreated?control?,respectively.Each group was further divided into subgroups according to drug treatment time,with 6 mice in each subgroup.CPT-11 was administered intraperitoneally at day 0.Afterwards,the mice continued to be farmed and their body weights were evaluated every day.The mice were sacrificed at day 3,day 7,day 14,day 28 and day 56,respectively.The peritoneal exudate cells?PECs?of each mouse were collected and counted,respectively.After labeling with antibodies,their phenotypes and numbers were further examined by flow cytometry.The surface molecule markers for mouse peritoneal macrophages were CD11b,F4/80 and MHC II,and that for B cells were CD23 and CD19.2.In Vitro:The PECs were seeded in glass-bottomed dishes?5×10⁵ cell/dish?.After 2 h incubation at 37?in humidified incubator of 5%CO₂,unattached cells were discarded.The large peritoneal macrophages were evaluated by their expression of surface markers?CD11b and F4/80?and intracellular transcription factor GATA6.3.Cell adoptive transfer:PECs and bone marrow cells?BMCs?from syngeneic healthy mice were transferred into the peritoneal cavities of the mice that had been treated with CPT-11?200 mg/kg body weight for once?7 days before,respectively.The peritoneal macrophages and B cells in these mice were detected at day 28 by flow cytometry.4.Bacterial Infection in Mice:Mice were divided into 3 groups:Control,Vehicle and CPT-11 group.The Control group received no treatment,the Vehicle group were peritoneally injected with vehicle solution,and the CPT-11 group was intraperitoneal-ly injected with 200 mg/kg of CPT-11.After 7 days,the mice of all the three groups were peritoneally injected with a sublethal dose?1×10⁹ CFU?of viable E.coli suspe-nsion.Observations were made every 6 hours and the survival of the mice was record ed for a total of 96 hours.5.Histochemical observation:After the mice were sacrificed,the whole intestine-s were taken and were immediately fixed in 4%neutral formalin solution.Paraffin-e mbedded sections were treated by hematoxylin-eosin staining and were observed and recorded with an inverted Zeiss microscope with Zeiss Axio Observer D lens and a co lor CCD camera.Results:1.After intraperitoneal injection of CPT-11 in the mice,the large peritoneal macrophages?LPMs?with a phenotype of CD11b⁺F4/80^hiGATA6⁺were disappeared within 1 week.But after 14 days,the numbers of LPMs in the peritoneal cavities were gradually increased.However,the recovery rate was slow,and two months?day56?after CPT-11 treatment the number of LPMs was only about half as compared to that of normal control mice,indicating that CPT-11 intraperitoneal injection has obvious toxic effects on the peritoneal macrophages and the recovery process was long.2.After intraperitoneal administration of CPT-11,the numbers of B1 and B2cells were significantly eliminated.At day3 after CPT-11 treatment,the percentage of B cells in the peritoneal cavity was reduced to 3%of the total number of the PECs.But after 56 days,the number of B cells in the peritoneal cavity was almost completely recovered to the normal level of control mice.3.After CPT-11 treatment,the mice received syngeneic PECs and bone marrow cells?BMCs?at day 7.The phenotypes of the PECs were analyzed 21 d after the adoptive transfer.The result showed that the adoptive transfer with PECs significantly increased the ratio and number of LPMs(CD11b⁺F4/80^hiMHC?^low)in CPT-11-treated mice as compared to that control group?CPT-11 only?.Transfer with BMCs also slightly increased the number of LPMs as compared to that of CPT-11 group,but the differences were not statistical significantly.Besides,the ratios and numbers of SPMs were not significantly changed by the adoptive PECs and BMCs.4.Subsequently,we observed whether CPT-11-treatment impaired the innate immune function against bacterial infection.Mice were first intraperitoneally injected with CPT-11 and 7 days later their peritoneal cavity were infected with viable E.coli.CPT-11 treatment significantly reduced the mouse survival rate as compared to control or vehicle.5.Oral administration of CPT-11 also eliminated the PECs and made the intestinal tissue injured.Oral administration of CPT-11 twice?at day 0 and day 1?at the dose of 400 mg/kg for each time in C56BL/6 mice did not significantly reduce the percentages of peritoneal macrophages?LPMs and SPMs?and B cells at day 3.Nor did it change the percentages of GATA6-positive cells in the peritoneal macrophages.However,oral CPT-11 resulted that the total number of PECs was reduced about 1/3?from¹.5×10⁶ to⁹.5×10⁵ for each mouse?.Moreover,oral CPT-11 induced severe epithelial vacuolation in the full-length of the intestines and the mice began to die from day 4 and on.In another experiment,the drug was administered orally at the same dose only once?at day 0?.The total number of PECs in the CPT-11 group were decreased to⁹×10⁵?¹/3 reduction as compared to control?at both day 7 and day 14.These results suggest that the impairment of oral CPT-11 on the intraperitoneal innate immune cells was weaker as compared to intraperitoneal CPT-11,but oral CPT-11was more toxic to the intestinal epithelium.Conclusion:Intraperitoneally CPT-11 treatment induced the depletion of resident peritoneal macrophages?LPMs?and B cells,and the recovery process of these cells was slow.Adoptive transfer with syngeneic PECs into the CPT-11-treated peritoneal cavities accelerated the recovery of these innate immune cells,and such a method is expected to accelerate the recovery process of the innate immune cells?immune function?of the peritoneal cavity of chemotherapy patients.