Video: Lymph node histology

What do you like to do? Oh, I like dancing. And rock music? But not country. Hanging out with friends and… Meditating. And I also like going out. Going out and just hanging sometimes. Wow, me too! We’ve so much in common, eh?

Ever been speed dating? We all know that the odds of meeting the love of your life are not always that encouraging but many live in the anticipation that they will bump into the right person at the right time and find the one that fits them perfectly.

Lymphocytes in our body know this story all too well and spend a whole lot of their time running around looking for the one that is holding that very specific antigen which perfectly matches the antibodies on its surface. One place they hang out to do this is in our lymph nodes, where they go from cell to cell for hours, if not days, looking for that special one who holds the antigen which they are destined to bind with. It’s speed dating on steroids, except when they find their perfect match, they don’t exactly fall in love, but rather multiply creating more antibodies to kill said invading antigen and all of its family or get someone else to kill it for them. Think of it as the bachelor gone really, really wrong. So let’s see what these immune system speed dating buzz are all about as we explore the history of the lymph node.

So we’ve established that our main topic for today are lymph nodes. We will, of course, look at their general structure and gross anatomy but our main focus today is the microanatomy of these structures; in other words, their histology. Let’s have a little overview of the structures we’ll cover in this tutorial.

We’ll start by looking at the location and overall distribution of lymph nodes around the body. Next, we’ll look at the general structure or organization of a lymph node. We’ll then get into the meat and the potatoes of this tutorial by looking at lymph node histology. And on the histological slide, we’ll look at some of the gross anatomy structures we identified earlier as well as some other structures that cannot be identified from the macroscopic level. Then finally, we will as always finish up with some clinical notes.

Okay, guys, I hope you’re ready to learn about lymph nodes because we’re going to get into it starting with lymph nodes in the body.

Right, let’s start with the basics. What are lymph nodes and why do we need them? Well to answer this question, we need to look at the bigger picture; in this case, the lymphatic system. The lymphatic system has two main jobs. The first is to collect interstitial fluid, also known as lymph, that surrounds all of the cells of our body that will later be reintroduced into the bloodstream. The second job is to monitor and destroy pathogens by filtering this lymph.

The lymphatic system consists of the primary lymphoid organs, namely the bone marrow and thymus as well as various secondary lymphoid organs which include but are not limited to, the lymph nodes, spleen, mucosa-associated lymphoid tissue, tonsils (palatine and pharyngeal), and Peyer’s patches found in the small intestine. These are then all connected by lymphatic vessels which complete the framework of the lymphatic system. But today, you already know we’ll be focusing our attention on the makeup of a lymph node.

There are around 400 to 450 lymph nodes scattered all over the body, in particular, in areas vulnerable to the entry of pathogens. You can see the wide lymphatic network on the image on the screen. Each lymph node is basically a meeting point where different types of cells known as antigen-presenting cells, the body’s surveillance team, come to display information about foreign antigens which they have encountered around the body. These cells mingle with patrolling lymphocytes, also arriving at the lymph node, which have come to see if the unique antigen that they are designed to neutralize has entered the body. If they find their nemesis antigen, an immune response is mounted and an army of specific lymphocytes is created to combat the foreign antigen around the body.

Now that we understand the bigger picture, let’s look at the general organization of a lymph node. If you zoom right in, you’ll notice that it is roughly bean-shaped. On its convex surface, you’ll find afferent lymph vessels carrying lymph into the lymph node. On the opposite concave surface, we find blood vessels and efferent lymph vessels carrying lymph away from the node. This region from which the efferent vessels exit lymph node is called the hilum of a lymph node. The lymph node is divided into layers. From superficial to deep, these are the capsule, superficial cortex, deep cortex, and medulla. Let’s see some notable structures which we’ll discuss in this tutorial.

The subcapsular sinus is located just below the capsule. The capsule sends trabeculae into the parenchyma of the lymph node dividing it into lobules. In the cortex of each lobule, we find numerous structures known as lymphoid nodules. In the medulla, we find medullary cords and medullary sinuses, which are the spaces surrounding them. But there’s no point in just learning from pictures, is there? Let’s look at some real histological slides.

This is the histological slide we’ll be focusing on today. As you can see, it clearly has the same general shape as the image we were just looking at as they both show a sagittal section of a lymph node. We have this convex surface here where we find afferent lymph vessels entering the lymph node and the concave surface here is where we find the hilum of the lymph node. This is where efferent lymph vessels exit the node as well as the point of entry and exit of the lymph node vasculature.

The capsule covering the lymph node is composed of dense connective tissue mainly collagen but small amounts of elastic fibers and smooth muscle may also be present. It forms a thin layer of protection around the lymph node. Deep to the capsule, we find a space known as the subcapsular sinus, which receives the lymph from afferent lymph vessels, but we’ll discuss this in greater detail later on in the tutorial.

As we mentioned previously, this dense connective tissue of the capsule sends extensions from the internal surface of the capsule into the body of the lymph node forming the so-called trabeculae. The trabeculae form the basic divisions of the organ known as lobules and providing structural support for the blood vessels entering the lymph node. Smaller lymph nodes may have only one or just a few lobules with a small number of trabeculae, however, large nodes have much more trabeculae and contain many lobules.

We saw that a lymph node has plenty of functional components but without some sort of a structural network, we’d have a soup of lymphocytes on our hands. That is obviously not the case. Each lymph node is filled with a meshwork of reticular fibers formed by fibroblastic reticular cells which create a sort of scaffold giving the lymph node its structural integrity. In a histological slide, the fibroblastic reticular cells can sometimes be identified as large pale-staining cells among the darker more basophilic lymphocytes. The meshwork itself is largely obscured by the numerous lymphocytes which make up the bulk of the node parenchyma.

Moving inwards, we find perhaps the busiest layer of the lymph node which is known as the cortex. We say busiest because it is jam-packed with lymphocytes which circulate in and out of the lymph node as well as antigen-presenting cells, some of which present foreign material known as antigens for assessment.

The parenchyma of the lymph node is also filled with a meshwork of reticular fibers formed by fibroblastic reticular cells which create a sort of scaffold giving the lymph node its structure integrity. The cortex is actually subdivided into two regions – the superficial cortex, also known as the outer or nodular cortex, and the deep cortex, often referred to as the paracortex. Let’s take a closer look at these in our histological section.

We’ll start by looking at the superficial cortex which is arguably the most complex layer in a lymph node. First of all, how do you identify it? This is an easy question to answer. The superficial cortex contains rounded structures called lymphoid nodules or follicles which are only found in that layer. These nodules are basically aggregations of lymphocytes in one region as well as specialized cells known as follicular dendritic cells, also known as nodular dendritic cells. Now this is where things get a little more complicated because we need to understand the types and functions of the cells present in a lymph node.

The main types of cells present in the lymph nodes are lymphocytes which would come as no surprise since we just looked at them forming the lymphoid nodules. The two main types of lymphocytes found in the lymph nodes are T-lymphocytes or T-cells and B-lymphocytes or B-cells. Both types are produced in the bone marrow and can be further subdivided into various classifications of lymphocytes, but we won’t go into great detail about this today. The B-lymphocytes mature within the bone marrow whereas the maturation process for T-cells occurs in the thymus gland. This is why they’re called B- and T-lymphocytes; it’s based on their site maturation. The mature lymphocytes then enter the bloodstream and circulate around the body.

Let’s first talk about B-lymphocytes as this brings us back rather nicely to our superficial cortex because B-lymphocytes are the predominant cells of that layer. That means the nodules that we saw earlier are mainly clusters of B-lymphocytes. Inactive B-cells initially home to what are called primary lymphoid nodules which appear as a dark uniform cluster of small lymphocytes. If they encounter their antigen displayed on another cell known as a follicular dendritic cell, they are then encouraged to multiply, mature, and differentiate.

In contrast, if you look at this nodule here, you’ll notice that the center of it is actually lighter than the border around it. That’s because we’re now looking at a secondary lymphoid nodule. Here, B-cells begin to differentiate and the activated cells quickly proliferate filling the center of a nodule and pushing the quiescent cells to the periphery. These quiescent cells which become pushed to the periphery create a visibly dark border in secondary nodules called the mantle zone. It is full of small lymphocytes with large intensely staining nuclei which is why it appears darker. Other cells found in small quantities in the mantle zone include follicular dendritic cells, helper T-lymphocytes, and macrophages which we’ll speak more about in a few moments.

The area deep to the mantle zone in a secondary lymphoid nodule is called the germinal center which you can now see highlighted in green. The germinal centers might just be the busiest and most exciting part of the whole lymph node because this is where proliferation and differentiation of the activated B-lymphocyte is taking place. Germinal centers are organized into two distinguishable regions – a dark zone and a light zone. We say distinguishable because these two regions are not only distinguishable from each other in appearance but also because these regions are composed of B-lymphocytes at different stages of development.

We have memory B-cells which do a similar job to memory T-cells and initiate a much quicker response to familiar antigen. The other more common type of cell that a B-lymphocyte can differentiate into is a plasma cell. These cells attack antigens, which are basically any substances foreign to the body by secreting high levels of antibodies which either neutralize foreign invaders or take them for destruction by other types of immune cells like macrophages. Notes that the light zone is generally oriented towards the capsule while the dark zone is oriented towards the medulla.

The dark zone comprises of mitotic B-cells called centroblasts. These are basically B-cells that have begun proliferating and are actively multiplying. The centroblasts in the dark zone are not yet able to respond to antigens presented to them. The daughter cells of centroblasts in the dark area then move to the light zone and become known as centrocytes. At this point, these cells are smaller and have a cleaved nucleus compared to the centroblast precursors. They have stopped proliferating and they now have the capacity to bind antigen. This antigen is presented to centrocytes once again by follicular dendritic cells and it’s the presence of these cells with the centrocytes that gives this part of the germinal center a lighter appearance. If there is no new or ongoing stimulus, the proliferation of the centroblasts in the dark zone will subside after a couple of weeks with the dark zone continuing to reduce in size relative to the light zone.

You might wonder why you might want to present antigen to centrocytes at this stage. Well, the lymphocytes actually compete with each other for a chance to bind to the antigen presented to them and only the ones with the highest affinity for the antigen get to do it. The other centrocytes undergo apoptosis or programmed cell death. It may seem a bit grim and wasteful but this is a way to ensure that the B-lymphocytes that reached the final stage of maturation will do the most efficient job possible at recognizing and destroying pathogens. Macrophages specifically known as tingible body macrophages are also present and their job here is to clean up the apoptotic cells.

Okay, one last thing before we move on from the superficial cortex of the lymph node. Take a closer look at it. Do you see anything irregular about it? We know that the superficial cortex should be, well, superficial. But here lymphatic nodules normally only found in the periphery of a lymph node have spread throughout the lymph node. What we’re looking at here is a reactive lymph node with what’s known as follicular hyperplasia. This reaction causes nodules to appear in the deep cortex and the medulla instead of being limited to the outer cortex, and in turn, reduces the area the other structures occupy in the lymph node. Do you want to know exactly what condition we’re looking at? Stay with us until the end of this tutorial to find out.

We’re finally moving on to the layer of the lymph node deep to the superficial cortex called the deep cortex, also commonly known as the paracortex. It is characterized by an abundance of dark staining lymphocytes but lacks the nodules characteristic to the superficial cortex. Instead, it contains what are known as deep cortical units, which are located in darker staining areas of the cortex which have no nodules. Each lobule of the lymph node contains a single deep cortical unit which this time is populated by T-lymphocytes which just like B-cells proliferate when stimulated. To be more specific, we’re talking about helper T-cells, which present antigens to the other cells, and cytotoxic T-cells, which are the ones that attack and kill pathogens.

We have quite a unique feature in the paracortex called high endothelial venules. These venules are made up of cuboidal endothelium in comparison to the more typical squamous endothelium found in regular venules. These high endothelial venules allow free passage of lymphocytes from the bloodstream into the lymph nodes. You might wonder why this passage is important. Lymphocytes constantly circulate round our bodies in the bloodstream but stop off at various lymph nodes via these high endothelial venules to sample antigens picked by various types of antigen-presenting cells. One of these antigen-presenting cells which are found in the paracortex are interdigitating dendritic cells. These are not to be confused at all with follicular dendritic cells. They’re completely unrelated in terms of origin and function. They just happen to have a similar morphology to one another.

Since the amount of antigen in the blood is normally really small, lymphocytes would be unlikely to just bump into them. What happens instead is that these dendritic cells bind an antigen. They become activated and move into lymph nodes. They then present this antigen to T-lymphocytes in the paracortex of lymph nodes; quite a clever arrangement if you think about it. When there is a T-lymphocytes dominated immune response, the deep cortex can expand considerably; however, it will not form nodules like we saw in the superficial cortex.

That brings us to the deepest layer of our lymph node, the medulla. You can immediately tell it apart from the cortex because it doesn’t have the same densely packed arrangement of lymphocytes. Instead, the lymphocytes are arranged into medullary cords surrounded by medullary sinuses. Let’s take a closer look at these structures in our histological slide.

Now just like the deep cortex, the medulla becomes occupied by lymphatic nodules in the case of follicular hyperplasia but worry not, you can still identify it. Just look for areas which are not as dark and tightly packed, and that’s your medulla, okay? So what do we actually find in this region? Well, the medulla region is populated with plasma cells which are mature B-lymphocytes we saw earlier as well as T-cells and other B-cells. The lymphocytes in this region are arranged into medullary cords. These cords extend away from the deep cortex towards the center of the lymph node.

On histological sections, they appear as little dark clumps and clusters of small lymphocytes among the lighter medullary sinuses which contain fewer lymphocytes than the cords. Other types of cells found in the medulla are macrophages and fibroblastic reticular cells. Reticular cells produce reticulin fibers which form the reticular network supporting the architecture of every layer in a lymph node.

We have become quite familiar with our reactive lymph node but you must be itching to see a normal lymph node. We hate to disappoint so here you are. This is a little lymph node found next to the parotid gland. You can see that it has two very clearly defined regions – the darker outer region, the cortex, and the inner lighter stained region we know to be the medulla. Now let’s switch back to our much more exciting and familiar reactive lymph node.

Before we learn about the medullary sinuses, let’s take a step back and look at the circulation of lymph through the lymph node. The lymph enters a lymph node through afferent vessels at its convex surface, passes through a network of sinuses to then be collected at the medulla, and leave through the efferent lymph vessel at the hilum of the lymph node. Let’s take a closer look at the lymph pathway through a lymph node on our histological slide.

Starting at the outer convex surface of the lymph node, we find the afferent lymphatic vessels penetrating the capsule. They bring in lymph from the body into the lymph node. Afferent vessels have valves to ensure the unidirectional flow of lymph and you’ll find several of them along the convex edge of the lymph node. Each lobule of the lymph node will have one afferent vessel feeding it. Their walls are formed by a single layer of squamous epithelium surrounded by a layer of smooth muscle which by contracting allows the lymph to circulate around the body without a central pump. Finally, these vessels are covered with a layer of adventitia or fibrous connective tissue.

The afferent lymphatic vessels drain the lymph into a space between the capsule and the superficial cortex called the subcapsular sinus, sometimes known as the marginal sinus. In a lymph node, the reticular network forms the meshwork which is densely populated by lymphocytes. Where the lymphocytes are more diffuse, channels or sinuses are formed. These channels are lined by endothelium which allows free movement of lymphocytes and macrophages as lymph makes its way through a lymph node.

The circulation of lymph doesn’t end there. The lymph from the subcapsular sinus drains into trabecular sinuses, also known as radial cortical sinuses. The sinuses are found alongside trabeculae penetrating into the cortex and extending towards the medulla. The trabecular sinuses then transport the lymph into the medullary sinuses surrounding the medullary cords. Just like the other sinuses in the lymph node, the medullary sinuses are characterized by a reticular fiber network with some dispersed small lymphocytes.

Finally, the lymph from the medullary sinuses is collected into a single or a few efferent lymphatic vessels which then carry the lymph to be reintroduced into the bloodstream or to be filtered by other lymph nodes. Just like afferent vessels, these vessels have valves and the same three-layer structure of the vessel wall. These vessels exit at the lymph node hilum which is also the site where the artery and vein associated with the lymph node enter.

Alright, guys, that’s the histology of the lymph node covered. But remember the mystery condition affecting our lymph node from earlier? Should we find out what it is? What we’re looking at in this case is actually called follicular hyperplasia. It is the most common reactive change observed in lymph nodes causing enlargement of a lymph node due to an increased number of secondary nodules in the superficial cortex. There are various conditions which can affect lymph nodes, but there are several distinguishing features you can look for in a histological section to help you identify follicular hyperplasia.

You’d expect to see nodules of varying sizes with low density of nodules and large interfollicular areas. The nodules, in the case of follicular hyperplasia, don’t tend to extend beyond the lymph node capsule. Within nodules, you’d expect to see a well-defined mantle zone and polarization in the germinal centers, meaning you can tell apart the light and the dark signs. You’ve probably noticed that all of these features fit our histological section perfectly. So the big question is, what causes this?

It can be a symptom of several conditions, for example, rheumatoid arthritis, syphilis, Castleman disease, and other conditions, but it can often be a benign incidental finding of unknown etiology. It is quite common in children and teenagers. In these cases, it often affects a single lymph node and often resolves itself. It also occurs in grownups but in these cases, the involvement of several lymph nodes is more common and can result in the development of malignant lymphoma. Other conditions especially follicular lymphoma can mimic the symptoms of follicular hyperplasia so the treating physician may choose to monitor or to investigate the affected lymph node further and then prescribe appropriate treatment.

Alright, folks, that wraps up today’s tutorial, but before you run off, let’s summarize what we learned today.

We started by looking at the overall shape of a lymph node with afferent lymphatic vessels entering at its convex surface and efferent vessels leaving at the hilum at the concave surface. We then identified the capsule which sends extensions called trabeculae into the body of the lymph node and noted that the lymph node is formed around a meshwork of reticular fibers. Below the capsule, we saw the superficial cortex populated by lymphatic nodules. We identified primary nodules with the uniform appearance formed by quiescent B-lymphocytes.

The secondary nodules had two distinct zones. The mantle zone around the periphery was darker and contained inactive lymphocytes while the lighter germinal center was the site of active B-cell proliferation. The germinal center was further subdivided into a dark zone of mitotic centroblasts and a light zone where centrocytes were tested for the antigen affinity.

From here, we moved into the deep cortex which was largely populated by T-lymphocytes. We saw that it lacked nodules but contained high endothelial venules which allow the free movement of lymphocytes from blood into lymph nodes. And finally, we moved into the innermost layer, the medulla. We saw that the lymphocytes were much more diffused here and formed aggregations called the medullary cords. From there, we discussed the flow of lymph through a lymph node starting with afferent lymphatic vessels penetrating the capsule. The lymph then moved into the subcapsular sinus followed by trabecular sinuses, and finally, larger medullary sinuses surrounding the medullary cords. Here the lymph was collected and left the lymph node via the efferent lymphatic vessels at the hilum.

We finally looked at a condition called follicular hyperplasia and at how to identify it in histological section.

We hope you enjoyed this tutorial. See you next time and happy studying!