INTRODUCTION

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BLOOD PLASMA contains many factors that stimulate cellular proliferation, and it may contain some factors that oppose proliferation. Thus the effect of plasma on cellular proliferation probably depends on the concentration of these growth factors in plasma. Generally, plasma (serum) has a potent growth-promoting effect, and investigators often use blood serum as a nonspecific source of growth factors. However, cells within the extravascular space are exposed to interstitial fluid, not plasma or serum.

The growth factor concentrations in interstitial fluid may be much different from the concentrations in plasma. Normally, interstitial fluid is separated from plasma by the walls of the blood capillaries. The capillary walls may restrict growth factor movement between plasma and interstitial fluid (8). Furthermore, many factors are produced by cells within a tissue. The concentration of those factors may be very high in the interstitial fluid of the tissue in which the factors are produced, but their concentrations may be very low in plasma. On the other hand, some factors may be produced within the plasma. Thus the relative growth factor concentrations in interstitial fluid versus plasma probably depends on the production of the factors within the interstitial fluid or plasma and on the permeability of the factors in the capillary wall.

Several groups of investigators have studied the effect of interstitial fluid on cellular proliferation (4, 7, 9, 10). Because lymphatic vessels drain interstitial fluid from the tissues, the investigators have assumed lymph is equivalent to interstitial fluid. Thus the investigators collected lymph from animals or human subjects and tested the effects of the lymph on the proliferation of cells in culture. The investigators have variously concluded that the proliferative effect of lymph is greater (9), equal to (7), or less than (4, 10) the effect of plasma. However, most investigators have used lymph from the thoracic duct or other lymphatic ducts that drain lymph from several tissues. Consequently, it is impossible to relate the results of those studies to the proliferative effect of interstitial fluid from any specific organ.

In this study, we tested the effects of plasma and lymph from the lungs and intestine of sheep on the proliferation of fibroblasts we cultured from the sheep. We found that, compared with plasma, lymph was much less effective in stimulating fibroblast proliferation.

	METHODS

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We anesthetized four yearling sheep (40-52 kg) with thiopental sodium. Then we placed a tracheostomy tube and used a mechanical respirator to ventilate the sheep with 30% O₂ in room air. We placed polyethylene cannulas into the superior vena cava via a jugular vein and into the aorta via a femoral artery. We opened the right chest and ligated the distal tip of one lung lobe, and we removed ~1 g of lung tissue.

Once we had placed the vascular cannulas and taken the lung tissue samples, we cannulated lymphatic vessels from the lung and intestine. For the lung lymphatic, we cannulated a lymphatic vessel from the caudal mediastinal lymph node. To minimize nonpulmonary lymph flow to the node, we ligated and resected the tail of the node ~1 cm above the lower border of the pulmonary ligament (5). Then we opened the abdominal cavity and cannulated postnodal lymphatic vessels from the small intestine as previously described (6). To maintain lymph flow, we infused warmed Ringer solution intravenously into each sheep. Lymph samples were collected in test tubes containing EDTA. The lymph was centrifuged to remove white blood cells. To collect plasma samples, we opened the superior vena caval cannula and allowed the blood to drain into glass tubes containing EDTA. The tubes were centrifuged (10 min at 1,500 g), and the plasma was separated from the cells and frozen in plastic tubes. We used a refractometer (American Optical) to estimate the protein concentrations of lymph and plasma samples.

Cell culture. We cut the samples of lung tissue to ~1-mm cubes and placed the cubes in 60-mm plastic culture dishes with 5 ml of minimum essential medium (MEM; Life Technologies, Grand Island, NY), containing 10% fetal calf serum (FCS). After 7-14 days, the tissue was removed, and the fibroblasts remaining in the dish were allowed to grow to confluence. We used 0.25% trypsin and 0.05% EDTA to harvest the fibroblasts. Cells were used after the second to third passage.

The proliferation experiments. To test the effect of 10% lymph or plasma in MEM on cellular proliferation, we seeded ~1 fibroblast/mm² into 60-mm plastic culture dishes containing 5 ml MEM with 10% FCS. After 4 h, we removed the medium, washed the cells with physiological buffered saline, and placed 5 ml MEM containing 10% lymph or plasma with 7.5 U of heparin into the dishes. MEM with heparin, but without FCS or lymph or plasma, was used for control dishes. After 24 h, we placed the dishes onto an aluminum disk containing a 0.9-cm radius (area = 254 mm²) hole in the center. We counted the cells manually with a Nikon phase- contrast microscope through the hole in the disk. Only cells that adhered to the bottom of the dish were counted. We recorded that cell count as the initial number of cells. To quantitate the growth rate, we recounted the cells each 1-3 days for 6 days. To account for differences in the initial numbers of cells from dish to dish, we normalized the counts for each dish with the initial cell count for that dish. To compare the growth rates, we took the logarithm (base 10) of the normalized counts.

Statistics. Data are given as means ± SD in the text and means ± SE in Fig. 1. We used two-way analysis of variance to test for changes in the cell counts with time. To compare the cell growth rates between lymph or plasma samples, we subtracted the logarithm of the cell counts for each day and used two-way analysis of variance to test for differences in the data. We accepted P < 0.05 to indicate significant differences.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Cell counts over the 6-day proliferation period. N/N0, cell count/initial cell count.

	RESULTS

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We successfully cannulated lung lymphatics in two sheep and intestinal lymphatics in three of the four sheep. We took plasma samples from all four sheep. The protein concentrations for plasma, and lung and intestinal lymph were 4.7 ± 0.9, 3.3 ± 1.3, and 1.1 ± 0.5 g/dl, respectively.

The fibroblasts we cultured from lung tissue grew to confluence within 3-4 wk. We identified the cells as fibroblasts based on morphological appearance and the fact that fibroblasts readily grow from lung tissue. However, we cannot be certain that all of the cells were fibroblasts.

We performed seven proliferation experiments with MEM alone or MEM plus lymph and nine experiments with MEM plus plasma. The cells proliferated in MEM alone; however, the cell count on day 6 (the final day) was only 1.9 ± 1.6 times the initial count. Figure 1 shows the effects of plasma, lung, and intestinal lymph on cellular proliferation for all experiments. We found significant increases in cell counts for all groups. Cells in MEM containing plasma or lung or intestinal lymph all proliferated significantly faster than cells in MEM alone. Furthermore, cells in MEM plus plasma proliferated ~10-fold faster than cells in MEM plus lymph (P < 0.05). We found no significant growth rate difference for cells grown with lung versus intestinal lymph.

	DISCUSSION

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Our results demonstrate that lymph from the lungs or small intestine is much less effective than plasma in stimulating cellular proliferation. The weak effect of lymph (relative to plasma) is consistent with the hypothesis that lymph contains lower concentrations of growth factors compared with plasma. However, it is possible cells within the lung and intestine release factors that inhibit cellular proliferation. A higher concentration of inhibitors in lymph versus plasma could have caused the difference in cellular proliferation we found.

Our study is different from previous studies in several ways (4, 7, 9, 10). First, most other investigators have used lymph from the thoracic duct (4, 7, 10). Thoracic duct lymph is a mixture of lymph from most tissues of the body. Thus the effect of thoracic duct lymph on cellular proliferation may not reflect the effect of lymph from any specific organ. In contrast, we used lymph from the lungs and intestine, and we believe our data show the effects of growth factors in the interstitial fluid of those organs.

Although we attempted to collect pure lung lymph, lymph collected from the sheep caudal mediastinal lymph node contains some lymph from diaphragmatic lymph vessels that drain to the node (5). However, there is usually little flow through diaphragmatic lymph vessels in animals in which the diaphragm is not contracting (5). There was no diaphragmatic contraction in our anesthetized open chest sheep. Thus we believe the lymph we collected was at least 80% lung lymph (5). We believe the intestinal lymph we collected came only from the intestine because we traced the prenodal lymphatics directly to the intestine. Furthermore, the lymphatics drained from a segment of omentum that contained only intestine.

Another difference in our study and previous studies is that some investigators have used serum derived from lymph or plasma (3, 7, 9). That is, they allowed the lymph or blood to clot and then separated the serum fluid from the clot. Serum derived from lymph or plasma contains thrombin, and thrombin may stimulate cellular proliferation (2). Also, serum derived from blood usually contains growth factors released from activated platelets (platelet-derived growth factor). To prevent clotting and other calcium-dependent reactions, we collected our samples in EDTA. Then we centrifuged the samples to remove the cells and platelets. However, we cannot rule out growth factor release from the cells or platelets before we centrifuged the samples. Also, some platelets may have remained in the plasma after we centrifuged. If growth factors were released from the cells or platelets into the plasma, they could have increased the effect of the plasma samples on cellular proliferation.

Some investigators have tested the effects of lymph from one animal species on cells cultured from another species or on established cell lines (4, 6, 9, 10). The results of their studies may have depended on the cross-species activity of growth factors. In this study, we tested the effect of lymph and plasma from sheep on cells we culture from the same sheep.

Like other investigators, we assumed that the composition of lymph is similar to the composition of interstitial fluid. Unfortunately, this assumption may be incorrect because the composition of lymph may be modified as the lymph passes through lymph nodes (1). We could not collect prenodal lung or intestinal lymph in sufficient amounts to perform the proliferation tests of this study. Thus we cannot be sure the concentrations of growth factors in the lymph we collected equaled the concentrations in prenodal lymph or tissue fluid.

In conclusion, our results indicate that lymph from the lung and intestine is much less effective in stimulating cellular proliferation compared with plasma. This difference is probably due to a lower concentration of growth factors in lymph compared with plasma.