Why do anal wounds heal adequately? A study of the local immunoinflammatory defense mechanisms.
OBJECTIVE
The aim of this study was to identify the tissue defense immunoinflammatory mechanisms present in the healing process of anal region wounds resulting from hemorrhoidectomy by the open technique.


METHODS
Immunohistochemical techniques were applied to biopsies of anal wounds obtained on Day 0 and Day 6 after surgery from 20 patients with hemorrhoid disease to characterize and quantify macrophages, T and B lymphocytes, and natural killer cells in high-power fields (400x). These techniques were also used to identify cells showing immunoexpression of cytokines (transforming growth factor beta 1, transforming growth factor beta 2, transforming growth factor beta 3) and constitutive and induced nitric oxide synthase. Plasma cells were quantified on slides stained with hematoxylin and eosin and the presence of immunoglobulin G, immunoglobulin M, and immunoglobulin A secreting cells was investigated by direct immunofluorescence.


RESULTS
An acute nonspecific inflammation with no lymphomononuclear-plasmacytic component was observed on Day 0. On Day 6, an inflammatory cellular infiltration rich in macrophages and lymphoplasmacytic cells was detected, which documented the participation of innate defense mechanisms and the adaptive tissue response. On Day 6, the mean number of immunoinflammatory cells were as follows: macrophages (CD68+) = 190.3; macrophages (HAM56+) = 184.3; T lymphocytes (CD3+) = 59.6; T lymphocytes (CD45RO+) = 47.7; helper T lymphocytes (CD4+) = 89.2; cytotoxic T lymphocytes (CD8+) = 29.4; B lymphocytes (CD20+) = 64.4; plasma cells = 1.7; natural killer cells (NK1+) = 12.9. Macrophages (HAM56+ and CD68+) were present in significantly higher amounts than those of the remaining ones. B lymphocytes (CD20+) predominated over T lymphocytes (CD3+), although the difference between the two cell types was not significant. Participation of the humoral immune system was characterized by the presence of immunoglobulin G-secreting cells. The cellular immune system was characterized by the identification of T lymphocytes (CD3+ and CD45RO+), most of them belonging to the T helper cell subpopulation (CD4+). These predominated in a significant manner over cytotoxic T lymphocytes (CD8+). Natural killer cells were present in small amounts. There was immunoexpression of constitutive nitric oxide synthase on Day 0 and on Day 6. Induced nitric oxide synthase was not identified on Day 0 but was present on Day 6. Transforming growth factor beta 2 and transforming growth factor beta 3 were expressed in endothelial cells on Day 0 and on Day 6, and transforming growth factor beta was also expressed in macrophages, endothelial cells, and fibroblasts on Day 6. Transforming growth factor beta 1 and transforming growth factor beta 2 were expressed significantly in macrophages, whereas transforming growth factor beta 3 occurred at similar proportions in the three cell types.


CONCLUSIONS
The host developed locally innate and immunologic defense adaptive mechanisms. The predominant local defense response involved macrophages. Natural killer cells and immunoexpression of constitutive nitric oxide synthase in endothelial cells were components of the noninduced innate response. In the induced innate response, in addition to neutrophils, there were large numbers of macrophages that were the major cells showing immunoexpression of transforming growth factor beta and induced nitric oxide synthase. The adaptive immunologic response was characterized by T and B lymphocytes. Helper T cells and cytotoxic T cells predominated in the cellular immune response and cytotoxic T cells and natural killer cells were present in small numbers. Secretory immunoglobulin G plasma cells were present in small numbers as a component of the humoral immune system.
de Paula
Pedro Roberto
PR
Departamento de Medicina, Universidade de Taubaté, Taubaté, Brazil. boccaradepaula@horizon.com.br
Matos
Delcio
D
Franco
Marcello
M
Speranzini
Manlio Basílio
MB
Figueiredo
Florêncio
F
de Santana
Ivonete Cândida Barbosa
IC
Chacon-Silva
Marcos Augusto
MA
Bassi
Deomir Germano
DG
D000328:Adult; D000368:Aged; D001003:Anal Canal / Q000601:surgery*; D000704:Analysis of Variance; D016207:Cytokines / Q000276:immunology; D005260:Female; D006484:Hemorrhoids / Q000601:surgery*; D006801:Humans; D007124:Immunoenzyme Techniques; D007694:Killer Cells, Natural / Q000276:immunology; D008214:Lymphocytes / Q000276:immunology; D008264:Macrophages / Q000276:immunology; D008297:Male; D008875:Middle Aged; D019001:Nitric Oxide Synthase / Q000276:immunology; D018709:Statistics, Nonparametric; D014945:Wound Healing / Q000276:immunology*
D016207:Cytokines; D019001:Nitric Oxide Synthase