Eosinophil production

Eosinophil granulocytes are found in all vertebrates and they are usually associated with parasitic and immunological diseases. Much of the previous work on eosinophils has concentrated on their distribution and variations in number in blood and tissues. This has led to many suggestions as to their possible functions. Few experiments have been done to study the way in which they are produced, and little quantitative information is available either about the kinetics of eosinophil production or the ways in which they can be mobilised. Even less is known about these processes under pathological states. Without this knowledge it has not been possible to distinguish factors altering eosinophil production from those influencing their turnover. For these reasons, the first part of this thesis describes experiments which were designed to measure the kinetics of eosinophil production, mobilisation and turnover in normal and stimulated rats.
Rats were given single injections of 10,000 Trichinella spiralis larvae and the extent of labelling of eosinophils in bone marrow and blood was followed on autoradiographs in these stimulated rats, and in normal rats. In stimulated rats the bone marrow content of eosinophils increased and this resulted in a peripheral blood eosinophilia 2andndash;10 days later.
Three sequential steps in rat eosinophil development in marrow were defined: primitive dividing eosinophils, more mature dividing eosinophils and non-proliferating eosinophils. The diameter of eosinophils was greatest just before they divided, and there was no relationship between eosinophil size and their maturity. Other experiments supported the concept that eosinophils leave the marrow and mature in the spleen before release into the blood.
The percentage of eosinophils in bone marrow showed no diurnal variation in normal rats. In rats given larvae intravenously the percentage of marrow eosinophils increased 23andnbsp;h after stimulation. The peripheral blood eosinophil count rose 1andndash;2 days later, and was related in parallel to the bone marrow level. The eosinophil mitotic index rose by a factor of 2.6, and there appeared to be a maximum number of eosinophil mitoses 40andnbsp;h after stimulation. The proportions of primitive and more mature dividing eosinophils did not alter during the first 3 days, showing that all the eosinophils in the proliferative compartment were stimulated equally.
The eosinophil cell cycle parameters in normal and stimulated rats were calculated from measurements of the proportion of eosinophil mitoses labelled at intervals after single injections of 3H-thymidine. In normal rats there was a wide variation of eosinophil cell cycle times with a median of 30andnbsp;h. In stimulated rats this was reduced to a median of 9andnbsp;h with a narrow distribution of cell cycle times. The post-mitotic rest period (TG1) and D.N.A. synthesis period (TS) were shortened. It was calculated that the earliest recognisable eosinophils divided a total of 4andndash;5 times in normal rats, and in stimulated rats eosinophils divided a total of 9andndash;10 times. Despite the additional number of divisions in the stimulated rats, marrow transit times were unaffected as the cell cycle times were shorter in the stimulated rats. It was concluded that increased eosinophil production came about by additional divisions in the proliferative compartment, associated with shortening of cell cycle times.
Eosinophil production rates were calculated from measurements of the number and labelling indices of eosinophils in weighed quantities of bone marrow combined with the known eosinophil cell cycle times. o Production rates increased by a factor of 28, from 0.5andnbsp;andtimes;andnbsp;103 to 14.4andnbsp;andtimes;andnbsp;103 new eosinophils/mg bone marrow/h.
Eosinophil kinetics in the blood were studied by giving single injections of 3H-thymidine to normal or stimulated rats, and the number of labelled eosinophils in blood was measured subsequently. The interval between injections of 3H-thymidine and the appearance of labelled eosinophils in the blood (emergence time) was 40andnbsp;h in normal rats and 17andnbsp;h in stimulated rats, but neutrophil emergence time was not altered. This demonstrated that different factors regulated the emergence times of these two types of granulocyte.
An eosinophil releasing factor was sought in plasma removed 12andndash;24andnbsp;h after the injection of larvae. Six hours after injection of 2andnbsp;ml plasma the eosinophil count in the blood of normal recipient rats doubled. The eosinophil releasing effect of plasma was not removed by dialysis and was stable at andminus;20°C. This eosinophil releasing effect was not associated with a neutrophil leucocytosis. Damaged eosinophils and homogenates of lungs containing larvae injected 16andnbsp;h previously did not induce eosinophil release. Many stimuli which induce an eosinophilia 3andndash;6andnbsp;h after injection may act by producing 'eosinophil releasing factor' in blood as was demonstrated with larvae.
Eosinophil half-life in the blood of normal rats was 6.6andnbsp;h. Surprisingly, eosinophil half-life did not decrease 2 days after injection of the stimulus at a time when many eosinophils were moving from the blood into the tissues. It was concluded that there was an increase in the size of the intravascular eosinophil pool size. Blood eosinophil half-life was lengthened 5 days after injecting larvae at the height of the eosinophilia, and it was also increased in splenectomised rats 2 days after stimulation.
In another section of the thesis, work is described which centered on the processes involved in the initiation of increased eosinophil production. Studies on rats injected with larvae proved to be very useful for studying the regulation of eosinophil production, as the tissues containing the stimulus were separate from the responding marrow. The initial response to the stimulus was studied in the tissues, lymph and blood.
First, the inflammatory reactions induced by Trichinella spiralis larvae in lung, liver and muscle were examined histologically. Mast cell degranulation was followed by mononuclear cell and eosinophil accumulation. In other experiments, mast cell degranulation was not found to be essential for eosinophil production and intravenous injections of larvae coated with peritoneal macrophages produced a smaller eosinophilia than uncoated larvae. However, in lymph nodes stimulated by Trichinella spiralis larvae, large numbers of dividing large pyroninophilic lymphocytes were found which were labelled with 3H-thymidine. These lymphocytes were considered to be the progenitors of the thoracic duct lymphocytes which have previously been shown to induce eosinophils production.
A study was mace of the distribution and fate of large pyroninophilic lymphocytes derived from the thoracic duct of rats with oral trichinosis. Large pyroninophilic lymphocytes, which were labelled with tritiated nucleic acid precursors, were distributed in a similar way in normal and stimulated rats. The majority of transferred labelled large pyroninophilic lymphocytes were found in the small intestine, spleen and lymph nodes, where they divided after 24andnbsp;h. At 48andnbsp;h they had the morphology of plasma cells. Only a few labelled large lymphocytes reached the bone marrow. When 51Chromium labelled lymphocytes were injected, less than 0.5% of the injected activity was found in each femur 22andnbsp;h later. These results showed that if large pyroninophilic lymphocytes initiate eosinophil production by short range processes, then these lymphocytes may be a small subpopulation of the cells normally found in thoracic duct lymph.
In related work, presented in the appendix, experiments were done to determine whether thoracic duct lymph, draining from rats with unilateral pyelonephritis, would stimulate neutrophil production when transferred to normal rats. No effect was found. This result, which agrees with other recently reported experiments, is evidence that neutrophil production is not initiated by lymphocytes.
It was concluded that in rats given Trichinella spiralis larvae intravenously, alterations in the numbers of eosinophils in the blood take place in several ways; either through changes in the distribution of mature eosinophils, or by variations in eosinophil production. The measurements which were done on each of these processes has shown that they are regulated by separate mechanisms, each with a distinct time course involving both humoral and cellular mediators. In conditions associated with an eosinophilia these mechanisms act in unison to provide eosinophils in increased numbers in response to the stimulus.
This work has shown that regulation of eosinophil production and blood eosinophil levels takes place by a number of inter-related processes, in ways which are as finely balanced as those known to control neutrophil or erythrocyte production and distribution.