The Kinetics of Mast Cells and Eosinophils in the Liver of Experimentally Infected with Schistosoma mansoni
Parviz Kermanizadeh PhD*, Paul Hagan PhD**, David W.T. Crompton PhD***
*Division of Immunology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran, **Division of Infection & Immunity, Joseph Black Building, IBLS, ***Division of Environmental & Evolutionary Biology, the Graham Kerr Building, IBLS, University of Glasgow, Glasgow, UK.
Objectives-
To study the livers
from S. mansoni infected mice during
16 weeks of infection with regard to granulomas and the
kinetics of mast cells and eosinophils in the granuloma.
Methods-The tissues were stained with a
newly developed method designed to detect mast cells and
eosinophils simultaneously.
Results- Granulomas appeared from the
sixth post-infection week and both the number and size of
the granulomas increased gradually in the acute phase of
the infection and then reduced as the infection matured.
Eosinophils comprised 97% of the cell types in the
granuloma and probably play an important role in
schistosoma egg destruction. At all times during the
infection mast cells comprised approximately 3% of the
cells in the granuloma.
Conclusion-The appearance of mast cells in
the granuloma may be dependent on factors other than
those associated directly with the granuloma.
Keywords • Mast cells • eosinophils • granuloma • schistosoma
Introduction
Adult Schistosoma mansoni which inhabit the inferior mesenteric veins, deposit embryo containing eggs. These eggs move into the intestine, but some are transported to the liver, predominantly lodging in portal areas, and others are carried to other organs where they become entrapped. These eggs are responsible for the major symptoms of schistosomiasis. The toxic egg material destroys the host tissue cells and the antigenic material stimulates the development of large inflammatory reactions (granuloma) around the egg material.
This granuloma is considered to serve as a protective barrier by sequestering the toxic and antigenic substances secreted continuously from the schistosoma eggs, thereby, preventing further diffusion of these harmful materials.
On the other hand, granulomas may replace the damaged parenchyma of the organs in which they lie by producing and setting down collagen between the normal cells and converting the functional tissue into fibrotic tissue and initiating periphlebitis and fibrosis in the liver.1,2 Portal hypertension, splenomegaly, ascites and esophageal and gastric varices develop due to the resulting impaired blood circulation.
Eosinophils are a known source of cytokine production.3-6 The important role of these cells in host defence mechanisms against parasites is well established.7 By producing cationic and major basic proteins, eosinophils have cytotoxic effects on parasites. Mast cells also produce a large variety of chemicals and cytokines and may play a very important role during the processing of the granuloma.
Materials and Methods
A total of 68 female outbred CFLP mice, each weighing between 30 g and 37 g, were infected cutaneously with 100 cercariae of a laboratory-maintained Puerto Rican strain of Schistosoma mansoni after inducing anesthesia. During a 16- week period, four to five mice were processed each week for histologic studies. Liver tissue was fixed in MT fixative for 24 h at room temperature, and then tissues were processed and embedded in paraffin. Sections of 5 µm thickness were cut and stained by a new method.8 The area was calculated for each granuloma section. At least ten granulomas were measured and subjected to cell counting for each sample of liver tissue. The number of mast cells and eosinophils were counted in each granuloma at x400 magnification and the mean number of all counted cells (per mm2) was plotted arithmetically. The number of granulomas with and without eggs and the total number of granulomas in a minimum of 10 graticules of each infected liver section were selected randomly and the mean number of each type of granuloma was computed in 100 mm2. The size of 10-20 eggs as an ellipse was calculated in each liver sample and their mean was computed.
Results
From the third post-infection week, eosinophils were observed in the liver samples, and by the fifth post-infection week, clusters of eosinophils could be seen, predominantly in peri-vascular areas of the liver tissue. No mast cells were observed at these times. At week 6, granulomas started to form and rapidly became prominent in tissue sections. The number of granulomas increased from 39 per 100 mm2 at week 6, to 309 per 100 mm2 at week 12, decreasing to 165 per 100 mm2 by week 16 which was the end of the experiment (Fig 1). The size of the granuloma also gradually increased from 0.018 mm2 to 0.037 mm2 at the 6th and 9th post-infection week respectively, and later decreased to 0.025 mm2 at the end of the experiment (Fig 1). As the number of granulomas increased, the number of those without eggs also increased. At week 12 (Fig 2), the number of granulomas with and without eggs reached 133 and 176 per 100 mm2 respectively. Only a few mast cells were observed in liver granulomas (Fig 3). Most granulomas were located around the perimeter of the liver. Compared with the number of eosinophils, mast cells comprised only a small percent of the total granuloma cell number in this experiment. Mast cells were observed in the liver tissue by the 6th post-infection week. The number of the mast cells increased from 23 per mm2 per area of the granuloma at the 6th post-infection week gradually reaching a maximum of 105 per mm2 area of the granuloma by the 9th post-infection week. The number of mast cells decreased to about 90 per mm2 at the end of the experiment (Fig 3). The number of eosinophils was seen to be high ranging from 3,299 per mm2 of the granuloma at week 6 to 4,545 per mm2 by week 8, eventually decreasing to 2,160 per mm2 at week 16 which was the end of our experiment (Fig 3). The size of the eggs in the liver tissue showed some fluctuations but seemed to reach a stable state during the chronic phase of infection (Fig 2).
Discussion
Hepatic schistosomiasis and the cellular organisation of schistosoma granulomas have been studied by many investigators.9,10 In general, T cells have been considered as the most important cells in the regulation of granuloma formation11,
especially during infection with Schistosoma mansoni.12 In addition, eosinophils constitute as much as 50% of the cells present in the granuloma13 and significant numbers of mast cells and their effects on granuloma formation14,15 have also been reported. According to the results obtained in the current study, the number of
granulomas increased from the 6th post-infection week to reach a peak by the 12th week. This peak may be the time of peak egg production by the adult S. mansoni female worms; granuloma numbers remaining constant thereafter. The size of granuloma varied with time, reaching a peak at
week 9, becoming smaller by week 16. This finding is in keeping with ideas on the down-regulation of the granulomatous response.16 Evidence of repair and healing of damaged tissue was also visible by week 16. The mast cell dynamics fit well with the increasing size of the granuloma. The number of granulomas without eggs peaked at the 12th post-infection week. This may have been due to the activity of eosinophil numbers which increased after week 8 and remained high throughout the experiment. Unlike eosinophils, mast cells increased in number from week 6, reaching a maximum of 105 per mm2 area of granuloma by week 9. At week 10, mast cell numbers declined to 90 per mm2 per area and remained at that level until week 16 (the end of the experiment), by which time 3% of the cells present were mast cells and 97% were eosinophils. Finally it seemed that the schistosoma eggs, or antigenic products derived from the eggs attracted eosinophils 17-19 rather than mast cells. It is reasonable to speculate from the complicated cell dynamics that mast cells probably did not contribute to egg destruction, but may have conversely played a central supporting role in granuloma formation. Mast cells were frequently observed near the middle or even towards the periphery of the cellular response producing the granuloma, but they were never observed inside or near the eggshell. One interpretation is that whilst the eosinophils may have an effector cell activity participating in egg destruction, the mast cell is more important in maintaining control of tissue repair and regeneration and may therefore have a subsidiary role in fibroblast activation, regulating their activity in collagen production.20 Fibrosis is probably of some value to the host since it serves to isolate the egg and it’s products from the remainder of the tissues. Fibrotic insulation of the granuloma occurred whether or not the eosinophils succeed in completely destroying the egg. However, when granulomatous fibrosis was evident, the number of eosinophils found in these granulomas was reduced to a few or even zero. Other changes were also evident. The staining properties of the granuloma altered, and became blue. Eosinophils could be observed outside the layers of fibrosis around the granuloma and the granuloma itself was largely devoid of cells with only a few mast cells in its middle or external layers. Only rarely were eosinophils found in contact with the granuloma at this stage. During the phase of acute inflammation, eosinophil and mast cell numbers increased to a maximum, by the 8th to 9th post-infection week, respectively. This increase was proportional to the increase in the size of the granuloma but was not proportional to the increase in the number of granulomas. This may represent a limitation on the capacity of the animals to produce these cell types during acute inflammation resulting from infection with S. mansoni. Several points can be gleaned from these studies. First, eosinophils are important in the destruction of the eggs of S. mansoni. Second, the observed increase in mast cell numbers always followed the increase in the number of eosinophils. Third, in the liver of mice infected with S. mansoni, the number of mast cells remained constant. These results are consistent with the view that mast cells serve as an accessory cell during infection with S. mansoni. Mast cells may play a dominant role in recruiting the inflammatory cells and they may take part in granuloma regulation by releasing histamine21 or producing cytokines.22,23 Fourth, in severe infections, as judged by the number of granulomas in the tissue, the number of mast cells are relatively lower than in light infections. Finally, the distribution of mast cells in S. mansoni infected mice seems to be independent of the parasite antigen and the inflammation itself. In the current experiments, despite finding high numbers of parasite eggs in the liver of infected mice, the number of mast cells in the liver was always much lower than that found in other tissues and in severe infections as judged by egg numbers. During these experiments, relatively fewer mast cells were found in tissues. On the basis of these observations, other factors appear to be important in the distribution of mast cells within the tissues. The antigen of the parasite may be a stimulus in mast cell production but it is of secondary importance in determining the final tissue sites occupied by mast cells.
References