Eicosanoid Cascade

Pathway 1: Immediate AA release and PG production

The most straightforward pathway for AA mobilization and prostaglandin production takes place immediately after the stimulus has interacted with its receptor. For the P388D1 macrophages this occurs when the cells are stimulated with zymosan. For PG production this involves the release of AA from phospholipids via a PLA2 and its conversion to PGs via one of the two known cyclooxygenases (COX-1 or COX-2). The process itself may last from minutes up to a few hours, and its distinguishing characteristic is that pre-existing enzymes mediate it. The phospholipases and cyclooxygenases involved are already present in the cells and are not synthesized de novo. The immediate pathway for AA mobilization and PG production operates in almost all cell types and is mediated by the early activation of the GIV PLA2. This PLA2 appears to be responsible for the release of the entire AA produced. There is no apparent involvement of the sPLA2 in this pathway. Since the GIV PLA2 requires an initial burst of Ca2+ to interact with its membrane substrate, this pathway is typically activated by short-term stimuli that induce transient increases in the intracellular Ca2+ concentration.

In most cases, the GIV PLA2 is coupled to the COX-1 for the production of prostaglandins. There are some cases, however, where COX-2 is also constitutively present in the cell. When this is the case, COX-2 also participates in the process.

Pathway 2: Primed immediate AA release and PG production

The primed immediate pathway of AA mobilization and prostaglandin production involves the sequential exposure of the cells to two different stimuli. The first one does not activate the cells itself but leaves them prepared to maximally respond to a second stimulation. This kind of response is typical of macrophages exposed to LPS. Scheme 1 represents our current understanding of the mechanism of the primed immediate pathway during inflammatory stimulation of the P388D1 cells.

LPS primes the cells thus preparing them for activation by inflammatory mediators such as PAF. The LPS priming step can be prevented by inhibitors of RNA transcription and protein translation, indicating that de novo protein synthesis is a required event. Thus, this pathway involves the participation of both constitutive and inducible enzymes. By stimulating protein synthesis, LPS increases the levels of PLA2 and COX activity in the cells, and this allows the cells to subsequently produce a larger response.

In the second phase of the primed immediate pathway, the primed cells are activated by PAF. Acting through a specific receptor, PAF produces at least two kinds of signal which, acting in concert, lead to the immediate activation of the GIV PLA2 in an intracellular compartment. It is now clear that one of these signals is the liberation of sequestered intracellular Ca2+.  The second signal remains undefined. However, we have found that PIP2 activates GIV PLA2 in vitro thus opening the possibility that PIP2 production constitutes the second signal.

The PAF signals accomplish two tasks. They initiate the rapid secretion of an sPLA2, which, once secreted, re-associates with the cellular surface. They also lead to the activation of the GIV PLA2 that produces a transient elevation of intracellular free AA levels. Elevated free AA during the initial stages of P388D1 cell activation may serve important metabolic and biochemical functions, one of which appears to be to help activate the GV PLA2 which is now found on the outside of the cell surface. The molecular mechanism involved in the activation of the outer surface GV PLA2 by AA derived from the GIV PLA2 is unknown. AA containing phospholipid must be transported to the external leaflet of the plasma membrane for the GV PLA2 to have access to it. Therefore membrane asymmetry and phospholipid transport across the membrane may play a role in activating this GV PLA2.  It is not clear whether PAF in any way directly activates the GV PLA2 or its expression in the cells.

Once the GV PLA2 is activated at the outer surface, it hydrolyzes phospholipids and releases AA. Part of this AA is released to the extracellular media while the rest is re-captured by the cells and made accessible to COX-2 for the generation of prostaglandins during cellular activation. We have evidence that the COX-1, the constitutively expressed enzyme that seems to be responsible for the production of the PG needed for maintenance functions, does not see this AA. It apparently obtains its AA from another source, perhaps from the GVI Ca2+-independent PLA2.

Enhanced mobilization of AA is followed by its enhanced conversion to PGs by either COX-1 or, if present, by COX-2. When both COX isoenzymes are present, whether one of them is utilized preferentially over the other appears to depend on cell type and stimulation conditions. In the P388D1 macrophage cell line exposed to LPS/PAF, most of the PG produced arises from COX-2, not COX-1, but this might not constitute the general mechanism.

An interesting feature of the primed immediate pathway is that the sPLA2 releases most of the AA under these conditions, not the GIV PLA2. However, the activity of the sPLA2 appears to be strikingly controlled by the GIV PLA2. This means, that, even though the GIV PLA2 is no longer the major provider of AA, it is still the key enzyme in the release due to its upstream position in the signaling cascade. How this happens is uncertain at present, but it appears that the GIV PLA2 directly regulates the mechanism of activation of the sPLA2 . Even though the sPLA2 contributes the majority of the AA acid, the GIV PLA2 still liberates AA that is converted to PGs. Therefore, the sPLA2 may be acting more as an enhancer rather than as an absolutely required component of the pathway.

Subsequent to our studies establishing the model depicted in Scheme 1, other research groups have recognized it as an applicable model for immediate PG production in activated mammalian cells. Our discovery that GV PLA2 is a novel and important effector for AA release, was confirmed by the groups of Herschman and Arm in murine mast cells. Moreover, the key discovery that there are two different PLA2s involved in the AA release, acting in different roles, but in an inter-related manner, has also been confirmed in a variety of other cell types and has decisively influenced further research in the field. Scheme 1 is now widely accepted as the model of PG production in cells of the immune system and may be applicable to other types as well.

Pathway 3: Delayed AA release and PG production

This pathway proceeds gradually for several hours and requires de novo synthesis of particular enzymes. This is the response elicited by LPS and other pro-inflammatory stimuli when incubated for long periods of time. PAF activation is not required for this process. AA release occurs only after a time lag of about 2-3 h, followed by an enhanced PG production. Delayed AA release has been correlated to the LPS-induced expression of sPLA2 (either GIIA or GV, depending on cell type), and PG production has been correlated with the induction of COX-2. Thus, increased synthesis of sPLA2 by the cells eventually results in large increases in free AA release. Part of this AA will be metabolized to prostaglandins by COX-2. As in the primed immediate pathway, in the delayed pathway the major provider of free AA is not the GIV PLA2 but the sPLA2. Yet, the sPLA2 activity is still controlled by the GIV PLA2. Thus, GIV PLA2 activation is again an important signaling step. We and others  have provided strong evidence that GIV PLA2 activation is required for the cells to synthesize new sPLA2 protein. This finding suggests that the GIV PLA2 can affect gene expression in addition to its direct role in AA release. Enhanced COX-2 expression at the mRNA level is another striking biochemical marker of the delayed phase, and also appears to be downstream of GIV PLA2 activation. It is important to note that in the delayed phase, COX-2 is always present and is solely responsible for the delayed synthesis of prostaglandins. COX-1, albeit present, does not participate in this process. Why this is so is unknown at present, but this appears to be the general mechanism.

Pathway 4: Ca2+-independent Immediate AA release and PG production

In our studies of the WISH cells, we have found a PMA stimulated pathway that is similar to the immediate release pathway described above. It does not require ansPLA2, LPS, or PAF, and it occurs in a short time period. It differed in one important way; it did not require a Ca2+ spike. This may represent a fourth pathway. We have found that a similar pathway is also present in the P388D1 cells.