Hyaluronidase (HYAL) induces an increase in leukocytes in bronchoalveolar fluid (BALF) of C57Bl/6 mice. (A) Total cells obtained from bronchoalveolar space after 4, 24, 48 and 96 h of inoculation with 4, 8 or 16 U of bovine testicular HYAL. Values represent means ± standard error of mean (SEM); n = 4; * P < 0.05 compared to phosphate buffered saline (PBS); ANOVA test was used. (B) HYAL induces almost exclusively mononuclear cell increases in the bronchoalveolar space after 96 h of inoculation with 16 U of bovine testicular HYAL in C57Bl/6 mice. N.d = not detected. Values represent means ± SEM; n = 4; * P < 0.05, ** P < 0.01 compared to PBS by ANOVA. (C) 16 U HYAL did not induce oedema after 96 h. The total protein content was measured in BALF. Llopopolysaccharide was inoculated intranasally (i.n.) as a positive control of oedema (500 μg/μL in 20 μL). Values represent means ± SEM; n = 4; P = 0.003 t test compared to the PBS. (D) Tityus serrulatus hyaluronidase (HYAL-TS) also induced mononuclear increase in the bronchoalveolar space after i.n. inoculation of 16 U in C57Bl/6 mice. N.d = not detected. Values represent means ± SEM; n = 5; * P = 0.003 using test t, compared to the PBS. (E) HYAL inactivation by heating reduces mononuclear cell numbers in bronchoalveolar space. 16 U of bovine testicular HYAL were heated or not at 60°C or 95°C i.n. inoculated and BALF cells collected 96 h later. Values represent means ± SEM; n = 5; * P < 0.001 compared to PBS; # P < 0.001 compared to active HYAL by ANOVA. (F) Photomicrographs of representative lung sections obtained from mice inoculated with PBS or HYAL. Tissues were stained with H&E to investigate inflammatory cells accumulation. Original magnification: 100x.