Inflammation is a biological response that protects the host against infection, promotes tissue repair and recovers tissue homeostasis. Understanding the mechanisms that regulate inflammation is essential, as inflammation can become a maladaptive response that leads to the development and exacerbation of many diseases. The cytokines of the Interleukin-1 (IL-1) family, namely IL-1a and IL-1b, initiate and propagate inflammatory responses. While IL-1a and IL-1b signal through the same IL-1 Receptor, they differ in their expression, protein interactions, subcellular distribution and secretory mechanisms. Research has widely focused in IL-1b secretion, while the mechanisms that regulate IL-1a function are poorly understood. The aim of this thesis was to investigate multiple aspects that distinctly regulate IL-1a secretion. Firstly, the work presented here shows that while IL-1a and other IL-1 family cytokines are secreted in absence of cell lysis, IL-1a has a distinct secretory pathway related to the proteases calpain-1/2 and it may be dissociated from membrane permeability in contrast to IL-1b. Secondly, IL-1a nuclear localization was found to inhibit IL-1a release and activation, and IL-1a pro-domain was found to be necessary for IL-1a stability and activation. Finally, a new method to screen the IL-1a interactome by enzymatic proximity labelling was set up, which will be used to find novel functions of IL-1a protein interactors. Thus, the findings in this thesis give new insights in the mechanisms that regulate IL-1a secretion, and contribute to the understanding of an inflammatory signalling pathway of therapeutic interest.