Severe hypoplasia of lymphoid tissues in Mo1 deficiency

Perspectives in Pathology Severe Hypoplasia of Lymphoid Tissues in Mol Deficiency HIROYUKI NUNOI, MD,*'~ YASUHIDEYANABE, MD,* SHIGENORI HIGUCHI, MD,* HIROYUKI TSUCHIYA, MD,* JIRO YAMAMOTO, MD,* ICHIRO MATSUDA, MD,* MAKOTO NAITO, MD,t KIYOSHI TAKAHASHI, MD,t KYOKO FUJITA, MD,w MASASHI UCHIDA, MD,w KUNIHIKO KOBAYASHI, MD,wMASAYOSHI JONO, MD,* AND HARRY MALECH, MD '~ A lymph node was examined I from a 4-year-old female child with documented "Mol" (CR3) deficiency. There was hypoplasia of the lymph node, with small, poorly delineated germinal centers and overall lymphopenia. Retrospective analysis of the clinical course of a deceased elder sister of the index patient indicated that the elder sister probably also had "Mol" deficiency. Review of findings at autopsy revealed severe hypoplasia of all lymphoid organs with lymphopenia. Our observations suggest that lymphoid tissue hypoplasia may be a feature of CR3 deficiency and is likely a result of the defective lymphocyte adherence functions to endothelium associated with absent lymphocyte functionassociated antigen (LFA-1). HUM PATHOL19:753--759. 9 1988 by

r e p o r t o f the histopathology o f l y m p h o i d tissues in patients with this disease. We f o u n d severe hypoplasia o f l y m p h o i d organs, with l y m p h o c y t e depletion in tissues obtained f r o m two siblings with r e c u r r e n t infection s y n d r o m e , o n e o f w h o m had d o c u m e n t e d Mol deficiency. We suggest that this disease is associated with defective l y m p h o c y t e h o m i n g processes to thymus, l y m p h nodes, and spleen as a result o f the missing a d h e r e n c e proteins.

W.B. Saunders Company.

REPORT OF TWO CASES

CR3 (C3bi receptor) deficiency, also called Mol deficiency, is an autosomal recessive disease characterized by granulocytosis, r e c u r r e n t infections, and impaired adhesion functions o f phagocytes.1 Phagocytes f r o m such patients do not react with monoclonal antibodies against the CR3 (anti-Mol, anti-OKM1, and anti-Macl), n o r do phagocytes or lymphocytes react with monoclonal antibody directed at the lymphocyte function-associated antigen (anti-LFA-1). 2 T h e biochemical basis o f this disease is that these patients are missing a family o f t h r e e m e m b r a n e glycop r o t e i n s (CR3, LFA-1, p150,95), which are hete r o d i m e r s with u n i q u e ot-subunits a n d a s h a r e d [3-subunit. 3-5 T h i s l e u k o c y t e - a n t i g e n c o m p l e x was d e s i g n a t e d C D w l 8 by the S e c o n d I n t e r n a t i o n a l Workshop on Human Leukocyte Differentiation antigens. 6 However, t h e r e has not been a detailed

A 4-year-old girl was seen by us because of severe recurrent infections from 3 months of age (referred to as patient A). She was the second child of healthy consanguineous parents. The patient was the product of an uneventful pregnancy and delivery. Her umbilical cord separation was not delayed. Her first infection was otitis media, which recurred frequently during the first year of age. A bacterial skin infection without pus formation was noticed at 6 months of age, and recurred for more than 1 year. She had five admissions for dehydration and severe stomatitis. She had brown hair (unusual in Japanese), numerous scars of the skin, cellulitis, chronic gingivitis, chronic rales and wheezing in both lungs, and hepatosplenomegaly. Her body height and body weight were within normal limits. Several lymph nodes were palpable in the neck, but her thymic shadow was not identified on chest x-ray. Laboratory tests performed at a time when she was not obviously infected were hemoglobin, 8.0 g/D1; RBC, 4,700,000/IxL with anisocytosis; and WBC, 28,400/pxL (basophiles, 1%; eosinophils, 1%; bands, 16 %; neutrophils, 57 %; monocytes, 4%; and lymphocytes, 21%). Her lymphocyte subpopulations were within the normal range (expressed as a percent of total lymphocytes: OKT3, 41.5%; OKT4, 24.5%; OKT8, 17.6%; OKT10, 60%; OKIA, 10.7%; B1, 12.7%; Leu-7, 12.2%). There was no detectable binding of anti-Mol (antiCR3 c~-subunit) or anti-NMA1/68 (anti-common [3-subunit) to any leukocytes. Total serum protein level was 8.0 g/dL. Increases in immunoglobulin (Ig) were noted: IgG, 2,346 mg/dL; IgA, 375 mg/dL; IgM, 325 mg/dL. The serum total hemolytic activity (CH50), and C3 and C4 levels were normal. Examination of a bone marrow aspirate revealed there were 120,000/txL nucleated cells (erythroblasts, 8.2%; promyelocytes, 2.4%; metamyelocytes, 1.2%; bands, 13.6%; segmented neutrophils, 44.8%; eosinophils, 10%; monocytes, 10%; and lymphocytes, 22.8%). Five minutes after

From the *Department of Pediatrics, tSecond Department of Pathology, and :~Department of Dermatology, Kumamoto University Medical School, Kumamoto, Japan; the w of Pediatrics, Yamaguchi University School of Medicine, Ube, Yamaguchi, Japan; and the 82 Disease Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infections Diseases,

Bethesda, MD. Revision accepted for publication February 22, 1988.

Key words: Mol deficiency, leukocyte adhesion, recurrent infection, lymphoid hypoplasia. Address reprint requests to Hiroyuki Nunoi, MD, Bacterial Diseases Section, LCI, NIAID, National Institutes of Health, Bethesda, MD 20892. 9 1988 by W.B. Saunders Company. 0046-8177/88/1907-002255.00/0

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HUMANPATHOLOGY Volume19, No. 7 [July 1988] injection of 0.03 mg of epinephrine, the number of peripheral blood granulocytes doubled from 23,000/IxL to 42,500/~L. The index patient's elder sister had also had recurrent infections from an early age (referred to as patient B). Patient B died of severe intractable pseudomonas pneumonia at 160 days of age, without a definitive diagnosis of the cause of her recurrent infection syndrome. The total leukocyte count in the elder sister's peripheral blood ranged from 27,900/mL to l l5,000/mL, with 75% granulocytes. Separation of the umbilical cord was not delayed in this patient. Lymphocyte subpopulations were normal, as were immunoglobulins and complement studies. An autopsy was performed, but the significance of the severe lymphoid tissue hypoplasia was not appreciated at that time.

sue blocks were recut and sections evaluated for this study using routine hematoxylin-eosin staining. RESULTS

Granulocyte and Monocyte Function in Patient A In vitro chemotaxis (under agarose) of granulocytes from patient A was 40% of normal (n = 4). In vivo chemotaxis (skin window) was markedly abnormal, with no emigration of granulocytes or monocytes evident at three, six, nine, and 12 hours, and only a few monocytes present at 24 hours. Superoxide production after activation with FMLP was normal. Phagocytosis by granulocytes from patient A was <10% of normal using opsonized Candida, baker's yeast, E coli, or S aureus (n = 3 for each). Adherence to nylon wool was 26% of normal (n = 3). Unlike normal neutrophils, most of patient A's neutrophils did not spread on plastic dishes.

MATERIALSAND METHODS

Cell Preparation Granulocyte and mononuclear cells were prep a r e d by the m e t h o d of Boyfim, with a slight modification. 7

FACS Analysis Granulocyte Function Assays In vitro chemotaxis was examined by the agarose plate method, 8 using N-formyl-methionyl-leucylphenylalanine (FMLP) as a chemotactic factor, and in vivo chemotaxis by the skin window method of Rebuck et al. 9 Adhesion was tested with nylon wool 1~ and plastic Petri dishes. 2 Phagocytosis was tested using fluorescein isothiocyanate (FITC)-conjugated Staphyloccus aureus, Escherichia coli, baker's yeast, and Candida albicans by the method of Oda et al,11 with a slight modification. Superoxide production after stimulation with FMLP was examined by the method of Nakagawara et al. ~2 Fluorescence-Activated Cell Sorter [FACS] Analysis With Anti-Mol and Anti-NMA1/68 Antibodies The blood cells were directly stained with FITClabeled anti-Mol and indirectly stained with antiNMAl/68 using FITC-labeled anti-mouse IgG antibody. Using the Ortho Spectrum III (Ortho, Hialeah, FL), granulocyte, lymphocytes, and monocytes were separated and the ratio of positive cells was calculated. Anti-NMA1/68 is a monoclonal antibody (similar to TS1/18, IB4, and 60.3) that reacts with the [3 subunit of CR3. t3 Histopathology of Lymphoid Tissues A cervical lymph node biopsy was obtained from patient A. In addition to routine hematoxylin-eosin staining, cryostat sections of patient A's lymph node biopsy were processed for immunohistologic staining using anti-IgG, anti-IgA, anti-IgM, anti-Leu-1, antiLeu-2a, anti-Leu-3a, and anti-Leu-12. At autopsy, patient B's lymph nodes, thymus, and spleen had been examined and tissue blocks prepared. These tis754

Binding of anti-Mol and anti-NMA1/68 to granulocytes and lymphocytes from patient A was reduced compared with controls (Fig 1). The decrease in monocyte binding was less striking than that seen in neutrophils. Histology and Immunohistology The cervical lymph node from patient A had a decrease in the number and size of germinal centers (Figs 2A and 3B) compared with a lymph node from normal age-matched controls (Fig 2C). In the index patient lymph node, there was some depletion of lymphocytes in the follicular area and paracortex. Histiocytes with weakly basophilic fine granules were abundant in the medullary and marginal sinuses, hnmunofluorescence labeling of sections of this lymph node from patient A using anti-IgG, anti-IgA, and anti-IgM indicated that small positively staining cells (plasma cells) were found in small groups scattered throughout the medulla (data not shown). Similar, though more striking, findings were seen in lymph nodes from a number of sites from patient B (Figs 2B, 2D, and 3C). At higher magnification of a cervical lymph node from patient B, it was noted that in the cortex there was a marked decrease in the number of high endothelial venules (HEV) and flattening of the HEV endothelial cells (Fig 3C). A similar, though less dramatic, flattening of H E V endothelial cells was also seen in the lymph node from patient A (Fig 3B). In order to determine whether recurrent infections alone might account for some of the findings, particularly the changes in HEV, we reviewed a cervical lymph node biopsy from a 4-year-old male patient with chronic granulomatous disease, who had an extensive history of recurrent life-threatening infections. A high-magnification photograph of the lymph node section from this chronic granulomatous disease

LYMPHOID HYPOPLASIAIN Mol DEFICIENCY[Nunoi et al] lymphocytes

granuJocytes

monocytes

5

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tology of these nodes was as noted above (Fig 2B). Similar findings were seen in mesenteric lymph nodes (not shown). The lymphoid structures of the appendix were also somewhat hypoptastic (Fig 2D). Microscopically, the thymus (Figs 2E and 2F) was also hypoplastic, and the basic structures of the medulla and cortex were indistinct. Lymphocytes were found scattered among thymic epithelia, and it was difficult to find Hassall's body-like structures. In the spleen (not shown), lymphocytes were greatly depleted and lymph follicles were rarely seen. DISCUSSION

Z

J .LZJ

Flow cytometry of granulocyfes, lymphocytes, and monocyte from patient A. Shown is direct binding of FITC-labeled anti-Mol and indirect binding studies with unlabeled anti-NMA1/68 followed by FITC-labeled anti-mouse IgG antibody, Fluorescence increases to the left on a logarithmic scale. The arrow in each panel is to the left of 99% of autofluorescence of cells in each study. Mo%positive granulocytes, lymphocytes, and monocytes comprised 1,4% 3.9%, and 33,2% in patient A, and 98%, 25%, and 90% in the control, respectively. NMAfl/68-positive granulocytes, lymphocytes, and monocytes comprised 28,4%, 2.4%, and 40.2% in patient A, and 96.5%, 95.7% and 88.9% in the control, respectively. FIGURE t.

patient is shown in Fig 3A. As reviewed above, this is contrasted at the same magnification in Fig 3B with a lymph node section from the patient with CR3 deficiency (patient A). The lymph node from the patient with chronic granulomatous disease is indistinguishable from a normal lymph node at the same magnification (not shown). In particular, there is normal cellularity, and the blood vessel contains normalappearing high endothelium. By contrast, the lymph node section from patient A is somewhat hypocellular, and the blood vessel endothelium is flattened. The autopsy-derived lymph node from patient B shows these changes to a much greater degree (Fig 3C). Figure 4 shows the immunohistologic staining of the patient A's lymph node. Leu-12-positive Blymphocytes were observed in mantle zones and germinal centers, but were weakly stained (Fig 4A). Leu1-positive T-lymphocytes were markedly decreased both in the medulla and the cortex (Fig 4B). The Leu-3a/Leu-2a ratio was normal (Figs 4D and 4C, respectively). Autopsy findings from patient B included enlargement of a few thoracic lymph nodes within which lymphocytes were depleted. The abnormal his-

The clinical course of patient A and reduced chemotactic, phagocytic, and adherence functions of this patient's phagocytes is consistent with CR3 deficiency. This diagnosis is confirmed by the marked reduction in binding of anti-Mol and anti-NMA1/68 to phagocytes from this patient. ~,~3 Since this disease is inherited as an autosomal recessive trait, ~4 it is likely that patient A's elder sister, patient B (who predeceased patient A) also had this disorder. Patient B's recurrent infection syndrome, associated with extreme granulocytosis, stongly supports this conclusion. Detailed observations of the histopathology of lymphoid tissues from patients with CR3 deficiency have not been reported. The one report relating to an appendix with chronic inflammation from a CR3deficient patient focused on the fibrosis and marked paucity of polymorphonuclear leukocytes, but did not comment on the characteristics of the lymphoid components of this tissue. ~5 In our studies, we focused on the histology of lymphoid organs. Biopsy of our index patient's cervical lymph node revealed severe hypoplasia associated with decreases in germinal centers and HEV, flattening of HEV endothelium, and a paucity of T-lymphocytes in the paracortex. Autopsy materials (thymus, peripheral lymph node, Peyer's patches, and spleen) from patient B showed similar but more severe hypoplastic findings and changes in endothelium. Recurrent infections alone are unlikely to have caused these specific findings, since these changes were not seen in a lymph node biopsy from a child with chronic granulomatous disease of similar age and similar infection history. In support of our observations in chronic granulomatous disease, Hotchi et al reported multiple suppurative granulomata in various organs, but did not describe any other histopathologic changes of thymus and lymph nodes in chronic granulomatous disease patients. 16 Since thymus hypoplasia was a striking finding at autopsy of patient B, we examined the thymus shadow on chest x-rays of patient A and two additional unrelated children with docur, ented CR3 deficiency. For comparison, we also examined chest xrays of three age-matched children with chronic granulomatous disease, another disease characterized by severe recurrent infections. The three chronic granulomatous disease patients had prominent thymus shadows on chest film, while two of the Moldeficient patients had no detectable thymus shadow 755

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FIGURE 2. Histopathologic findings in a biopsied lymph node and necropsied materials [see text]. A Cervical lymph node from patient A B Thoracic lymph node from patient B. C Lymph node from an age-matched control D Appendix lymph tissue from patient B, E Thymus from patient B. F Higher magnification of thymus from patient B.

and the third had a very small thymus shadow. These casual observations regarding x-ray findings are only suggestive, but considered with our histologic observations, indicate that evidence of lymphoid hypoplasia should be sought in other patients with Mol deficiency.

As lymphocyte recirculation between lymphoid organs and other tissues occurs, lymphocytes enter lymph nodes and Peyer's patches from the blood by specifically binding to the post-capillary HEV.17 The morphologic characteristics of HEV may depend on local T-lymphocyte traffic, since HEV flatten when 756

LYMPHOID HYPOPLASiAIN Mol DEFICIENCY[Nunoi et al)

FIGURE 3. Higher magnification of lymph node tissue showing high endothelial blood vessel from: (A) a patient with chronic granulomatous disease, (B) patient A, and (C) patient B.

T-lymphocytes are congenitally absent, such as seen with patients with severe combined immunodeficiency 18 or when T-lymphocytes are experimentally depleted. ~9 HEV regenerate when T-lymphocytes are repleted. 19 It is not clear whether vascular changes where lymphocyte infiltration occurs are the result or the cause of increased lymphocyte emigration at these 757

sites. Flattening of HEV and severe lymphoid hypoplasia in our patients could be a result of a reduction of reentry of lymphocytes into lymphoid organs (referred to as homing activity), though in vitro experimental evidence for abnormal homing activity by Mo 1-deficient lymphocytes has not been reported. Harlan et al 2~ and Jalkanen et a121 observed an

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FIGURE 4. Immunohistopathology of lymph node biopsy from patient ~ The cryostat sections were stained by the AvidinBiotin-Complex method, using the Leu-12 (A), Leu-1 (B), Leu-2a (C), and Leu-3a (D) monoclonal antibodies.

impairment of Mol-deficient neutrophils, monocytes, or lymphocytes to a d h e r e to h u m a n endotheliurn. However, these studies have not directly evaluated l y m p h o c y t e - H E V interactions. T o date, lymphoc y t e - H E V endothelial cell r e c o g n i t i o n mechanisms have b e e n assessed by d e v e l o p i n g m o n o c l o n a l antibodies against specific cell surface molecules o f lymphocytes (MEL-14, and H e r m e s - 3 ) and H E V endothelial cells o f the l y m p h n o d e ( H e r m e s - i ) . 22 Preliminary information indicates that monoclonal antibodies directed at CD 18 subunits and MEL- 14 antibody identify u n r e l a t e d molecules, t h o u g h both antibodies block T cell binding to high e n d o t h e l i u m . 2a However, m o n o c l o n a l antibodies directed at CD18 block a d h e r e n c e o f lymphocytes to a n u m b e r o f cell tvoes in a tissue nonspecific fashion, while MEL14 blocks o n l y l y m p h o c y t e b i n d i n g to h i g h end o t h e l i u m . 2~ I n t e r c e l l u l a r a d h e r e n c e m o l e c u l e 1 (ICAM-1) present on a n u m b e r o f cell types appears to r e p r e s e n t a l i g a n d f o r l y m p h o c y t e s u r f a c e LFA-l,23 and is d e m o n s t r a b l e on endothelial cell lines and largely evident only in inflamed tissue. 24 Such a non-tissue-specific accessory role for the CD 18 complex could account for the a p p a r e n t lymphoid hypoplasia in these patients r a t h e r than a speculation that the CD 18 c o m p l e x may have a specific biological m e a n i n g for l y m p h o c y t e - H E V binding. H a s k a r d et a125 and C a v e n d e r et a126 postulated that two d i f f e r e n t ligand.-receptor interactions may be involved in T cell-endothelial cell b i n d i n g (T-EC binding): an L F A - 1 - d e p e n d e n t process w h e r e b y l y m p h o c y t e s a d h e r e to u n s t i m u l a t e d EC, a n d an L F A - l - i n d e p e n d e n t process by which lymphocytes bind to I L - l - s t i m u l a t e d EC. O u r data does not di-

rectly address these processes. However, the LFA1 - i n d e p e n d e n t process might explain why some lymphocytes r e m a i n in these patients lymph nodes and why M o l - d e f i c i e n t patients a p p e a r able to m o u n t a delayed-type hypersensitivity response. H e n d r i k s et a119'27'28 observed the results o f occlusion o f the a f f e r e n t lymphatic vessels and conc l u d e d that m a c r o p h a g e s and T cells in l y m p h nodes may release mediators that activate endothelial cells o f H E V to p r o d u c e a secretary product. T h e latter may act as a r e c e p t o r on the H E V for recirculating lymphocytes. 29 Macrophages and T cells in CR3 deficiency are functionally abnormal, a~ It is possible that in vivo they might fail to regulate the influx o f l y m p h o c y t e s f r o m the b l o o d s t r e a m into l y m p h nodes. We c a n n o t tell if impaired decreased influence o f resident m a c r o p h a g e s and T cells on lymphoid tissue e n d o t h e l i u m , o r an i m p a i r e d l y m p h o c y t e endothelial cell interaction in M o l - d e f i c i e n t patients, is a cause o f the observed severe hypoplasia and lymp h o p e n i a o f l y m p h nodes. We c o n c l u d e f r o m o u r studies that l y m p h o i d h y p o p l a s i a m a y be o n e o f the h a l l m a r k s o f CR3 deficiency, and is consistent with [he fact that these p a t i e n t s c a n n o t r e s p o n d well to n e w a n t i g e n challenge. 31 F u r t h e r e x a m i n a t i o n o f the histology o f l y m p h o i d tissues in patients with CR3 deficiency, and additional in vitro and in vivo studies o f the mechanisms regulating l y m p h o c y t e h o m i n g to l y m p h o i d organs, may clarify the significance o f these histologic abnormalities.

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Acknowledgments. We are indebted to Professor Masaru Yoshinaga (the First Department of Pathology, Kumamoto University Medical School), Professor Masahiko Kotani (the Second Department of Anatomy, Kumamoto University Medical School), and Dr John Gallin (Director, Intramural Program, National Institute of Allergy and Infectious Diseases) for their valuable suggestions. REFERENCES

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