The Relationship Between Cytokine Regulation and Anti-Inflammatory Action of Amine-Carboxyborane in L929 Fibroblasts and IC-21 Macrophages

The amine-carboxyboranes anti-inflammatory agents were shown to block TNF α release at 90 min. and IL-1 release at 5 hr. from macrophages. The agenst competed with L929 fibroblasts high affinity receptors for endogenous cytokines which regulate the inflammation process. Blocking the TNFα receptor at 90 min. by the agents from 10 to 50  μΜ, resulted in lysosomal hydrolytic enzyme inhibition and lowering of prostaglandin synthesis as well as reductions in calcitonin high affinity receptor binding and calcium influx into the cells. IL-1 receptors when blocked by the agents at 5 hr. resulted in a reduction of NAG activity and leukotriene synthesis. An elevation of proline incorporation into collagen occurred at 90 min. and 24 hr. in the presence of the agents.


Introduction
Amine-carboxyboranes and related derivatives have been shown to be potent anti-inflammatory and antioosteoporosis agents in rodents I'4. These agents were shown to block septic shock, pleurisy, arthritis and induced edema in mice at doses from 2 to 8 mg/kgI. Previous studies have demonstrated that these agents reduced hydrolytic enzyme activities, in addition to prostaglandin and leukotriene synthesis in macrophages, leukocytes and Be Sal osteoporotic cells 3. Other studies have indicated in vivo and in vitro that the amine-carboxyboranes reduced TNF and IL-I levels after challenge with LPS 1,2 Cytokine release from inflammatory cells is related to the induction of fever, acute phase response and release of secondary chemical mediators, e.g. prostalandins and leukotrienes 5. IL-I is a pyrogenic and chemotaxic factoru.
Studies have demonstrated that TNF is maximally released from IC-21 mouse macrophages from 60 to 90 min whereas ILol is maximally released from P388D macrophages at 5 to 8 hrI.
IC-21 macrophages were selected because they release cytokine as well as hydrolytic enzymes and secondary chemical mediators of inflammation.
L929 fibroblasts were selected because they are inflammatory target tissue cells which contain high affinity receptors for cytokines and are responsible for wound repair after injury has occurred. TNF= release from ICo21 macrophages peaks at 90 min. and the effects of the agents from 12.5 to 50 M were determined at 90 min and 5 hr. IL-I release from P388 D macrophages was determined at 90 min. and 5 hr. The

Results
The LPS induced TNF release from IC-21 cells and IL-I release from P388DI cells was maximum at 90 min. and 5 hr., respectively. LPS concentration of 5, I0 and 20 g/ml of growth medium were examined in both assays and i0 g/ml afforded the best release of TNF at 90 rain and ILol at 5 hr. Thus, this concentration of LPS was selected for the following studies. The reduction of TNF release caused by aminecarboxyboranes from 12.5 to 50 M followed a concentration-response curve over at 90 mi.n. and 5 hr [ Table I]. It was interesting to note that the agents still suppressed TNF= release from ICo21 macrophages at 5 hr. long after the peak release of the cytokine induced by LPS.
The effect of amine-carboxyboranes on the release of IL-I from P388DI cells using I0 g/mi LPS at 90 minutes and 5 hours was reduced at all concentrations of agents, but most significantly at 25 and 50 M (43-100%) [ Table 2]. FTrthermore, after 90 minutes in the presence of 50 M of Compounds #i and #3, and after 5 hours with 50 M of Compound #3, IL-I release was inhibited 100%. The agents were able to suppress IL-I release from P388DI cells at an early time i.e 90 min., when IL-I levels were normally substantially lower after LPS induction [  ..0 rain hrs -m-ln ?5-.rs acontrol=91.2 ng IL-I released;DControl=166.8 ng IL-I released;*p <0.001 TNF high affinity receptor binding on L929 cells was reduced with amine-carboxyboranes at i0 BM from 30 to 120 rain. This is the same period of time when the maximum release of TNF occurs from IC-21 macrophages. Higher concentration of the agents after 90 min incubation did not improve the inhibition of TNF= high affinity binding to L929 fibroblast high affinity receptors [ Table 4].    ,ound, The amine-carboxyboranes at 12.5, 25 and 50 pM significantly decreased IL-I high affinity binding of IC-21 receptors at 90 rain. at a time when time when maximum binding occurs in these fibroblasts [ Table 7]. At 12.5 BM the maximum reduction of IL-I high affinity binding was Vol. 2, No. 5, 1995 The Relationship Between Cytokine Regulation and Anti-Inflammatory Action of Amine-Carboxyborane There was essentially no effect of amine-carboxyboranes at 12.5, 25 and 50 BM on NAG activity in IC-21 macrophages at 90 minutes or 5 hours [Tables 8 and 9].   Prostaglandin cycloo-oxygenase activity in IC-21 cells was measured in the presence of amine-carboxyboranes at 12.5, 25 and 50 M after 90 minutes and 5 hours. Prostaglandin cyclo-oxygenase activity was reduced at all times in IC-21 macrophages. In IC-21 cells, prostaglandin cyclooxygenase activity was maximally reduced by Compound #I at 90 minutes (  5. 1995 The Relationship Between Cytokine Regulation and Anti-Inflammatory Action of Amine-Carboxyborane At 90 minutes incubation the magnitude of reduction of 5' lipoxygenase activity in IC-21 cells was less with Compounds #2 and #3 which achieved a 33-39% reduction at 50 M (Table 16) than at 5 hours incubation, when the 5'-lipoxygenase activity was reduced 46-52% by all three of the amineocarboxyboarnes [ Table 17].  (Table 18). At all times, the calcium uptake was decreased significantly and at 5 hours the uptake of calcium by L929 cells to reach its lowest level for each compound.  Pure recombinant human TNF or amine-carboxyboranes were added to L929 cells and the effects on proline incorporation into collagen and noncollagen were compared for 48 hr. Proline incorporation into collagen was enhanced by the amine-carboxyboranes at 90 min. and at 24 hr. above control values. At 8 hours, Compounds #I and #3 caused a 32-34% reduction in proline incorporation into non-collagen of L929 cells. A moderate increase was observed at 90 min. Compound #2 was not effective in reducing proline incorporation into non-collagen of L929 cells at any time [Table 20]. TNF at I0 or I00 ng/ml of medium did not affect the incorporation of proline into collagen of L929 cells; however, proline incorporation into non-collagen was reduced 24% at i0 ng but not at I00 ng/ml at 90 min.
incubation.  (Table 21). Compound #I caused a 31-33% reduction of incorporation of proline at all concentrations of drug and was the most effective agent.
Compound #3 caused a 22-28% reduction at all concentrations and Compound #2 was not as effective producing a 14% reduction at 50 M. Vol. 2. No. 5. 1995 The Relationship Between Cytokine Regulation and Anti-Inflammatory Action of Amine-Carboxyborane  fibroblasts from 30 to 120 rain. The I0 BM concentration of the agents appeared to afford the best response in displacing the radioactive TNF which suggest at this concentration the agents were competitive with the cytokine's binding to this L929 protein receptor.
This receptor is important in the release of hydrolytic enzymes and proteolytic enzymes which initiate and cause tissue damage to tissue. The amine- The present study demonstrated that the time frame for the reduction of acid phosphatase activity in the presence's of the agents is consistent with the time of maximum inhibition of TNF high affinity receptor binding. The inhibition of TNF= receptor binding also correlated with the agents' ability to suppress the activity of the regulatory enzyme for prostaglandin synthesis at 90 min. Both the inhibition of hydrolytic enzyme activity and prostaglandin cyclo-oxygenase activity continued through 5 to 18 hours. Similar observation have been made when amine carboxyboranes were incubated with bone UMR-106 cells in that these same biochemical parameters were first suppressed at 90 min. consistent with suppression of the TNF receptor but continued for a much longer period of time than when peaked binding occurred with this cytokine receptor7.
TNF release was 120 ng/ml at 90 min the eak but was still at 80 ng/ml after 50 hr. well above background levels!. NAG hydrolytic activity was not inhibited until 48 hr. 5'-Lipoxygenase activity was suppressed after 5 hr rather than 90 min. The inhibition of this enzyme activity by the agents correlated more with the observed suppression ILol high affinity receptor binding than with suppression of TNF= high affinity binding. Similar pa.rallels were observed when UMR-106 bone cells were examined for effects of amineocarboxyboranes on cytokine high affinity receptors 7. Cytokines which are known to play a role in inflammation are TNF, ILol, 11-6 and IL-8. Apparently TNF effects are early, e.g. 30 rain to 6 hr., this is followed by II-i effects which begin around 2 hr and continue for 12 hr.
ILo8 is released late and has its high affinity binding to its receptors from 24 to 48 hr. Differences in response to the agents as well as the high affinity receptors for cytokines involved calcium uptake in L929 cells and bone UMR-106 cells Calcium uptake in bone cells peaked at 90 min and was apparently suppressed by TNF or the agents binding to TNF high Vol. 2. No. 5. 199 The Relationship Between Cytokine Regulation and Anti-Inflammatory Action of Amine-Carboxyborane affinity receptor at 90 min. but at 5 hr. this process was reversed and calcium uptake into bone cells was increased two fold at 5 to 8 hr 7. In L929 cells calcium uptake peaked at 5 hr. but was suppressed by the agents from 90 min to 8 hr. The agents suppressed dihydrovitamin D 3 binding to its nuclear receptor early but in bone cells increased the binding at 8 to 24 hr. which correlates with the increased uptake calcium into bone cells.
Calcitonin high affinity binding was not affected by the agents in a manner which would indicated it affects calcium uptake. In vivo the amineocarboxyboranes cause an elevation of PTH and 1,25-dihydrovitamin D3 after 14 days administration which correlated directly with an increase in serum calcium levels 2. Proline incorporation into collagen on the other hand. peaked in bone cells at 5 to 8 hr. while in L929 cells it peaked at 24 hr. in the presence of the agents. This process is required by both types of cells to increase bone tensile strength and to repair wounds. Thus, there does appear to be some tissue difference in response to the agents and the time of their maximum effects in a given type of cell.
Since the amine-carboxyboranes are very potent anti-inflammatory agents I4, the fact that they are able to block TNF, ILol and IL-8 receptor binding acting as antagonists would certainly explain the ability of the agents to reduce the inflammation process.
The early events of the inflammation process are probably mediated by invading macrophages and PMNs. These cells possess high affinity cytokine receptors which regulate the release and synthesis of hydrolytic and proteolytic enzymes which cause local tissue damage, vaodilation, generation of free radicals, infiltration of white blood cells, local heat, septic skock and anorexic wasting.
The latter stage involves tissue repair conducted by fibroblasts which also possess high affinity receptors for cytokines.
Both types of cells, macrophages and fibroblasts, contain high affinity receptors which regulate cellular events, their own release and the release of other cytokines 5,6. This is a chain reaction over time; thus, it is important that an effective anti-inflammatory drug block sequel events in the reaction as well as stimulate the repair process.
The amine-carboxyboranes appear to achieve these goals and further investigation as potential therapeutic agents is warranted.